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Gel Electrophoresis Size Marker
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Gel Electrophoresis Size Marker
Gel Electrophoresis Size Marker
Gel Electrophoresis
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Detergents
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Immunoassay Buffer
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Kontaminationen durch Nukleinsäurendurch Nukleinsäurendurch
Probleme & praktische Lösungen NukleinsäurenProbleme & praktische Lösungen Nukleinsäuren
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aus Chemie & Biologie& Biologie&
AppliCationsDer Anteil von molekularbiologischen Nachweismethoden ist in den letzten Jahren erheblich gestiegen, besonders in den Bereichen Qualitätskontrolle, Forensik, klinischer Forschung und Diagnostik – insbesondere der Infektionsdiagnostik. Gerade für diese Applikationen werden hochsensitive und gleichzeitig zuverlässige PCR-Tests benötigt. Dafür bietet AppliChem nun optimierte PCR-Kits an und widmet sich explizit der Hintergrundproblematik, die durch DNA belastete Reagenzien und Arbeitsplätze entstehen kann.
Nr.3AppliCations
Nr.3AppliCationsDNA-freie Reagenzien und Mastermixe für die PCR
AppliCationsAppliCations
Freie Nukleinsäuren verursachen als Kontaminationen große Probleme im Forschungs- und molekularbiologisch-analytischen oder klinisch-diagnostischen Labor. Durch die extrem hohe Sensitivität von DNA-Nachweistests, können kleinste Verunreinigungen in PCR-Ansätzen zusätzliche Arbeit bedeuten und im schlimm-sten Fall Ergebnisse verfälschen. Mit Derma-ExitusPlus™ (HHDK) aus der Serie von ExitusPlus™-Produkten wird erstmals ein völlig neuer Anwendungsbereich erschlossen bzw. zusätzliche Kontaminationsquellen ausgeschlossen.
Eine der Hauptquellen für Kontaminationen mit Nukleinsäuren ist der Experimentator selbst. Die Nukleinsäuren stammen z.B. aus Hautschuppen, Haaren und Speichel oder von Mikroorganismen, die seine Haut besiedeln oder z.B. beim Niesen freigesetzt werden. Gelangen diese in die PCR-Ansätze oder PCR-Reagenzien, können sie entsprechend der eingesetzten Primer (besonders 16S rDNA für Bakterien) leicht nachgewiesen werden. Ausserdem besteht die Gefahr, dass beim Öffnen und
Nr.5AppliCations
Nr.5AppliCationsDekontamination der Haut und Hände von Nukleinsäuren
AppliCations AppliCationsSize-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significantly slower through the column than larger molecules. Not to be mixed up with gel electrophoresis, there are big differences in terms of theseparation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.
It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to the longer path the smaller molecules must travel. The longer path arises from the pores of the beads. The smaller molecules can
Keywords
No.6AppliCations
No.6AppliCationsSize-Exclusion Chromatography for purification of biomolecules
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Agarose-Gel-Elektrophorese AppliCations
Using ready-to-use ELISA kits from manufacturers is easy and convenient. Sometimes however, home-made ELISA is required because there is no kit available with the right antibodies or the characteristics of the available kits such as their limits of detection are not appropriate. Ready-to-use ELISA kits from good suppliers mastored for two years at 4°C without any problem. With home-made ELISA it is a completely different story. For any new measurement one has to coat a new plate, because after storage of some days the plates don’t perform as well as before.
Why is there such a great difference in storage between home-made ELISA and ELISA kits?The reason is that in professional ELISA kit production the plates are not only blocked after coating, but also stabilised. This easy to perform process has been an industry standard for thirty years. For stabilisation of a plate one has to coating stabiliser solution. It is just as simple as a “second blocking step”. But there were no such high lutions freely available in low volumes for use in research lab until now. AppliChem now offers a product for use in every researchin volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easy-to-use and has a great advantage compared to almost any stabiliser used in industry. It gives better storage stability for coated antibodies and antigens than most other products do. And there is a second
Two benefits with one solutionWhen antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or inactive. Only around 2–8 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate, which can really differ from batch to batch or even from well to well.These differences from well to well can affect the variability of an assay, because the antibodies can be affected. If there waway of refolding antibodies and of preserving antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding of antibody conformation of some of the coated antibodies and 2. Preserving correct conformation during storage. These benefits are used for production of high-quality ELISA kits as well as in research applications now. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2–8 %. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to “Guidance for Industry: Bioanalytical Method Validation”, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays.The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwich ELISA with a monoclonal antibody has been
Keywords
Immunoassays
Antibody Stabilisation
ELISA Plates
Cross-reactivity
Interfering effects
Improving quality of ELISA
AppliCationsDie moderne Gentechnik zeigt, dass in vielen Fällen schon freie DNA-Moleküle für Infektionen, Rekombinationen oder biologische Transformationen ausreichen [1,2]. Zusätzlich werden die Nachweisverfahren für DNA-Moleküle immersensitiver. Daher wird die Detektion von Kontaminationen oder die Verhinderung von Amplifikations-Artefakten in der PCR für die Gentechnik, die Kriminalistik, die Biomedizin und die Hygiene immer wichtiger. Die vollständige Dekontamination von Geräten und Materialien von DNA-Molekülen wird so zu einem entscheidendenFaktor für die allgemeine biologische Sicherheit.
Alles oder Nichts: Erstaunliche ErkenntnisDas Mittel der Wahl zur Beseitigung von Kontaminationen durch NukleinDas Mittel der Wahl zur Beseitigung von Kontaminationen durch NukleinDas Mittel der Wahl zur Beseitigung von Kontamina säuren ist immer noch Chlorbleichlauge („bleach“) – ein Mittel das alles zerstört, nicht nur die Nukleinsäure. Dies hat uns veranlasst in Kooperation mit multiBIND Biotech, Köln, nach einer unschädlichen Alternative zu suchen und die molekulare Wirkungsweise der auf dem Markt befindlichen sonstigen DNA-Dekontaminationsmittel zu untersuchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Über-
suchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Über-suchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Überschuss) mit definierten DNA-Kontaminationen die Eigenschaften der konventionellen Mittel verglichen. Zwei Probleme werden offensichtlich: Erstens werden durch die konventionellen Mittel in keinem Fall die DNA-Moleküle effizient zerstört und zweitens enthalten diese Mittel Komponenten mit stark korrosiven oder giftigen Eigenschaften. Als Fazit daraus hat sich für uns die Notwendigkeit der Neuentwicklung einer effektiven Lösung zur DNA-Dekontamination ergeben, die wir hier als DNA-ExitusPlus™ und Autoclave-ExitusPlus™ vorstellen. Im Vergleich zu den herkömmlichen Produkten wird DNA und RNA schnell und effizient zerstört, ohne dass das Reagenz korrosive oder giftige Eigenschaften aufweist.Bei der DNA-Dekontamination unterscheidet man nach der molekularen Wirkungsweise der eingesetzten Mittel drei Grund-prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Denaturierung und Degradation.
prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Denaturierung und Degradation.
prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: ModifikaJe nach Zusammensetzung der Mittel können diese drei Prinzipien einzeln oder in Kombination angewandt werden.Da nach den aktuellen Erkenntnissen zum biologischen Risikopotenzial von freien DNA-Molekülen für eine wirklich sichere DNA-Dekontamination die Zerlegung dieser DNA-Moleküle in möglichst kleine Fragmente die wirkungsvollste Methode ist, wurden die gängigen konventionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest ver-tionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest ver-tionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest verglichen. Der DNA-Degradationstest erlaubt einen sensitiven, quantitativen Vergleich der Geschwindigkeit des DNA-Abbaus (Abb. 1 und 2).
Unerwarteter Weise haben wir festgestellt, dass einige der bekannten kommerziellen Mittel nur mit dem Prinzip der Modifi-kation oder Denaturierung der DNA-Moleküle arbeiten. Eine Zerlegung der DNA-Stränge erfolgt dabei nicht, sondern die genetische Information, für die diese DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine genetische Information, für die diese DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine genetische Informa
chemische Demaskierung der DNA-Moleküle durch Entfernung der blockierenden Gruppen würde die genetische Information wieder lesbar und am-plifizierbar machen. Nach dem heutigen Wissensstand zur Gentechnik und der Problematik der Neukombination von Erb-trägern sind solche Mittel eigentlich nicht mehr zeitgemäß. Aber auch die Mittel, die zu einer nachweisbaren Degradation
Keywords
Nukleinsäure-Dekontamination
DNA-DegradationstestPCR-Test
Autoklavieren von DNA
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Nr.1AppliCationsNukleinsäure-Dekontamination mit der ExitusPlus™-Technologie
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TransferMembranes
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SizeMarker•©2010AppliChem
Ever since the foundation of the firm, AppliChem has
investedextensivelyincommunicationandmarketing.The
freshandunusualappearanceattractedgreatattentionin
themarketfromtheverybeginning.Ourgrowthconfirms
therelevanceofthesemeasures.
Weofferourcustomersanextensivelibrary,withnumerous
brochuresandapplicationswhoseusemakeseverydaylife
inthelaboratoryeasier.
A comprehensive catalogue of products, with detailed
information provided by no other catalogue in the field,
isavailableinGermanandEnglish.
AppliChembringsadvantagesthroughknowledge.
“Thinkingwithoutknowledgemakeschancetheruler.”WernerKollath,Germanbacteriologist
©2010AppliChem•SizeMarker 1
contents 1 DNAmarker 2
1.1 Tips on the use of DNA length markers 2
1.2 AppliChem DNA marker overview 4
1.3 DNA marker 5
2 Dyesfornucleicacids 16
2.1 Overview 16
2.2 Methylene blue 16
2.3 Ethidium bromide 18
3 Proteinmarker 19
3.1 AppliChem protein marker overview 19
3.2 Precision protein markers 19
3.3 Prestained protein markers 21
4 Dyesforproteingels 24
4.1 Overview 24
4.2 Coomassie stain 24
4.3 Proteo-Dye 26
2 SizeMarker•©2010AppliChem
Tips on the use of DNA length markers
Correct storageDeproteinisedandlyophilisedDNAsamplesareextremelystable(>5years).ProblemsdonotusuallyoccurunlessDNAmarkersinsolutionarestored(>6weeks)atroomtemperature,theybecomecontaminatedwithbacteria,orarefrequent-lythawedandrefrozen(>20times).AfterdissolutionoftheDNA,wethereforerecommendaliquotingoftheDNAmarker in ready-to-usealiquots forstorageat -20°C(for2-4years).At+4°C,markers insolutionarestable forseveralweeks orevenmonths.Here,however,thereistheriskofbacterialornucleasecontamination.Thiscanbepreventedbystorageat-20°C.
End-labelingThankstotheirdeproteinisedandlyophilisedform,theseDNAmarkersaresuitableforuniversaluseforend-labelingwithmodifiednucleotides.LabelinginfourpositionsviatheterminalEcoRIgeneratedrecessedendsispossible,especiallywiththeDNAladder100bp(A3470)andtheDNALadderMix100-5000(A3660).ForthelabelingoftheDNA,theproductissimplydissolvedinTEbufferorbidistilledwater.
DNA staining with methylene blueOccasionally,ethidiumbromideisnotsuitableforstainingDNA.Analternativeismethyleneblue.Insuchcases,however,itmustbekeptinmindthatthemassofDNAusedmustbeincreasedbyabout30%andthatabout1.5hmoremustbeplannedfordestainingsteps.WithamethyleneblueconcentratesuppliedbyAppliChem,youachieveverygoodresults,evenforsmallfragmentsofabout200bp.
Mass calculations for single fragmentsUsingDNAmarkersofadefinedorigin,thecalculationofthemassofsinglefragmentsisrelativelyeasy.Theamountloadedperlane,e.g.1µg/10µl,isdividedbythenumberofbasepairsoftheDNAusedandismultipliedbythefragmentsize.Forexample:the267bpfragmentofthepBR322HaeIIImarkerwithaloadingamountof1µg:1µg:4361bp(pBR322)=0.229ng/basepairx267bpofthefragment=61.2ng/267fragment.Assuming comparable staining (saturationwithdye), themassofunknown fragments canbedetermined in thisway. Ifrequired,concentrationgradientscanbeloaded.
Less is often moreThedifferentgelformatsforagaroseandpolyacrylamidegelelectrophoresisandthevaryingsensitivityofstainingordetec-tionmeanthatitisonlypossibletogiveanapproximationoftherecommendedDNAamounttobeloaded.MostDNAmar-kersshowthebestseparationwithloadingamountsof0.5-1µgonagarosegels.Thegeneralruleis:thelowerthenumberofmarkerbands, the smaller the total amountneeded.A low loadingamount is alsoof advantagewith shortmigrationdistancesorverysensitivedetection.Whenusingpolyacrylamidegels,generallyonlyabout1/5-1/2theamountrequiredforagarosegelsisnecessary.ForDNAmarkerswithlargefragmentsandahighmass(e.g.equimolarmixturesorlambdaDNAmarkers),dependingontheagaroseconcentration,aminimumseparationdistanceisrequiredtoseparateorcompletelydistinguishbetweenthedominantupperbandsintermsofwidth.Thisseparationdistancecanoftenbereducedby10-15%byreducingthemarkeramount.Byusingequalizedmarkers,inadditiontoamarkedreductionofthemarkeramount,theseparationdistancecanbereducedbyafurther10-15%,frome.g.70to50mm,onastandardformatgel,toobtainthebestresults.
i n t r o d u c t i o n
1.1
©2010AppliChem•SizeMarker 3
How to achieve results quicklyEspeciallydeproteinisedDNAlengthmarkerscanbeseparatedverywellusinghighvoltages,e.g.within20minuteswithamigrationdistanceof70mmona1.2%agarosegelat120Volt.Thiscanonlyberealized,however,withahighqualitygelmigrationbuffer(thewaterqualitymustbe takenintoaccount!)andanadequateamountofbuffer(400-600ml).Poorbufferqualityresultsinoverheatingoftheagarosegelathighervoltagesandthisthereforecausesproblems.Ahighbuffervolume(thebuffercanusuallybereused4-6 times inoneweekwithoutproblems) leads theheatoff,provided thegelchamberforsubmersionisofadequatesize.
Staining front too intense?Insomecases,userswhoregularlyperformassayswithourproductssometimescommentthatthedyeconcentrationinthegelloadingbufferwesupplyistoohigh(onlylyophilisedmarkers).Ifthisisthecase,simplydissolvetheDNAlengthmarkerindoubleconcentrationinthegelloadingbufferandfurtherdiluteitwith1/2volumeTEbuffer.Evenwhenusinga50%solution,therearenoproblemswiththeresulting7%glycerolconcentrationtoincreasethesolutiondensity.
Plausibility of the results and ion concentrations:Non-plausible results (e.g.unexpected fragment runsat500bp insteadof350bp)mayoccurunder relatively extremeconditions(separationofrestrictionorPCRsampleswithaveryhighsaltcontent>150mM)oratveryhighvoltageswithrelativelyshortseparationdistances.Toidentifyerrors,1/10volumerestrictionbuffercanbeaddedtothealiquotoftheDNAmarker,forexample,ortherestrictionsamplecanbeappropriatelydilutedwithgelloadingbuffer(usually1/2volume).Inmanycases,loweringthevoltageduringseparationisalsohelpful.TheaccuratedeterminationofthesizeoffragmentsorPCRproductsmayalsobeimpaired,whensaltfrontscombinedwithlocalproblemsofleadingoffheatwithconductingawayheatandveryhighelectrophoresisvoltages,resultinpartialdenaturationoftheDNAfragments.TheseareconditionsthattheoreticallymayalsooccurwhenprocessingsamplesfromMaxam-Gilbertsequencingincombinationwithsamplebufferscontainingformamide.
i n t r o d u c t i o n
4 SizeMarker•©2010AppliChem
Prod. No. DNA Marker Bands Fragment Sizes [bp]
A3470 DNALadder100bp(lyophilised) 11 1000 900 800 700 600 500 400 300 200 150 100
A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100
A3302 DNALadder100bpequalized(lyophilised) 11 1000 900 800 700 600 500 400 300 200 150 100
A5216 DNALadder100bpplus 11 1500 1000 900 800 700 600 500 400 300 200 100
A3660 DNALadderMix100-5000(lyophilised) 17 5000 4000 3000 2500 2000 1500 1000 900 800 700 600 500 400 300 200 150 100
A3982 DNALadder250bp(lyophilised) 16 8000 6000 5000 4000 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250
A8640 DNALadder250bpplus(lyophilised) 15 12000 10000 8000 6000 5000 4000 3000 2000 1750 1500 1250 1000 750 500 250
A2667 DNALadder1kb(lyophilised) 11 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 500
A5207 DNALadder1kb 13 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 750 500 250
A6430 DNALadder1kbconcatamer(lyophlised) >25 1000 bpsteps(1000-approx.25000)
A7215 DNAMarkerquick-run(lyophylised) 5 2500 2000 1500 1000 500
A7222 DNAMarkerquick-runextended(lyophylised) 9 6000 4000 25000 2000 1500 1000 500 200 100
A4406 DNAMarkerpBR322-HaeIII(lyophilised) 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8
A5229 DNAMarkerpBR322-HaeIII 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8
A6927 DNAMarkerpBR328Mix(lyophilised) 12 2176 1766 1230 1033 653 517 453 394 298 234 220 154
A5194 DNAMarkerPhageLambdaStyI 11 19329 7743 6223 4254 3472 2690 1882 1489 925 421 74
A4412 DNAMarkerPhageLambdaBstEII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 BstEII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 Bst
A5220 DNAMarkerPhageLambdaBstEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 BstEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 Bst
A5589 DNAMarkerPhageLambdaHindIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 HindIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 Hind
A5223 DNAMarkerPhageLambdaHindIII 8 23130 9400 6557 4361 2322 2027 564 125 HindIII 8 23130 9400 6557 4361 2322 2027 564 125 Hind
A5235 DNAMarkerpUC19-MspI 12 501 489 404 331 242 190 147 111 110 67 34 26
A3996 DNAMarkerpUC19-MspI(lyophilised) 12 501 489 404 331 242 190 147 111 110 67 34 26
Lyophilised markers
Starting materialTheDNAinourlyophilisedlengthmarkersisamplifiedinlow-nucleasehostbacteria,restrictedwithqualityenzymes,andisdeproteinised.Thisensuresthattheycontainhigh-quality,low-proteinornuclease-freeproductswithverygoodmigrationpropertiesonagarose,agarandpolyacrylamidegels.
Quality controlNumerous gel separations of undigested plasmids and digested fragments formpart of themanufacturing process. Thefragmentmassisdeterminedusingtwocalibratedphotometersviaconversion(1OD260nm=50µgdsDNA)andisaliquotedat100%(+2%).EachbatchofDNAandgelloadingbufferissubjecttoafinalqualitycheckusinggelelectrophoresis.
StabilityLyophilisation of the DNA markers results in very stable products, without any significant impairment of quality atroom temperature after transport (even at the height of summer,> 35°C for several days) or after long storage times(>4yearsat-20°C).
ResuspensionBeforeuse,lyophilisedDNAmarkerscanbedissolvedingelloadingbufferoroptionalforend-labelinginsterile,bidistilledwater. The lyophilised form allows you the highest degree of flexibility (labeling, concentration, intensity of stainingbands).
Legal noteTheDNAladdersweremanufacturedbydigestionofplasmidswithEcoR I.Theplasmidsarelegallyprotected.Toproducecopiesapplyforapermissionfromthemanufacturer.
selection guide
©2010AppliChem•SizeMarker 5
selection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guide
) 11 1000 900 800 700 600 500 400 300 200 150 100
A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100
) 11 1000 900 800 700 600 500 400 300 200 150 100
A5216 DNALadder100bpplus 11 1500 1000 900 800 700 600 500 400 300 200 100
) 17 5000 4000 3000 2500 2000 1500 1000 900 800 700 600 500 400 300 200 150 100
) 16 8000 6000 5000 4000 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250
A8640 DNALadder250bpplus(lyophilised) 15 12000 10000 8000 6000 5000 4000 3000 2000 1750 1500 1250 1000 750 500 250
) 11 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 500
A5207 DNALadder1kb 13 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 750 500 250
A7222 DNAMarkerquick-runextended(lyophylised) 9 6000 4000 25000 2000 1500 1000 500 200 100
III(lyophilised) 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8
III 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8
A6927 DNAMarkerpBR328Mix(lyophilised) 12 2176 1766 1230 1033 653 517 453 394 298 234 220 154
I 11 19329 7743 6223 4254 3472 2690 1882 1489 925 421 74
EII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117
selection guideEII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117
selection guideEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 selection guideEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 selection guideIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 selection guideIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 selection guideIII 8 23130 9400 6557 4361 2322 2027 564 125 selection guideIII 8 23130 9400 6557 4361 2322 2027 564 125 selection guideI 12 501 489 404 331 242 190 147 111 110 67 34 26selection guideI 12 501 489 404 331 242 190 147 111 110 67 34 26selection guideI(lyophilised) 12 501 489 404 331 242 190 147 111 110 67 34 26
selection guideI(lyophilised) 12 501 489 404 331 242 190 147 111 110 67 34 26
selection guideDNA marker
DNA Ladder 100 bp (lyophilised) A3470DNAsizestandardformedium-sizedfragmentsandPCRproducts
Description Thefragmentsofthissizemarkeroccurinequimolarproportions,i.e.alsoallpossiblemarkingpositionsontheterminalEcoRIgeneratedrecessedends.Thebandmassincreaseswithfragmentlength.TheDNA isdeproteinizedand lyophilized.Thismarker isparticularly suitable forcom-parisonswithmedium-sizedDNAfragmentsandPCRproducts.The500basepairbandmasshasbeendoubledforeasierorientationon1.5-2.0agarosegels.Thesizeandmassofplasmidin-ser-tions or PCR products can be determined precisely in the range of up to 1000 base pairs atintervals of 100basepairs. An additional bandwith150basepairs hasbeen included for thedeterminationofsmallerPCRproducts.Thebestresultsareachievedafteramigrationdistanceofapprox.70-80mmonagarose gels. Each laneof anormal sized agarose gel (approx.80ml)
shouldbeloadedwithapprox.0.4-0.8µgofmarker.Loadingbufferissuppliedseparately.
Number of bands 11
Fragment sizes (bp) 1000,900,800,700,600,500(x2),400,300,200,150,100
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Storage -20°C
Loading volume 0.4-0.8µgperlane
Package size A3470,0050 50µg
1.21.21.21.21.21.21.21.2) 11 1000 900 800 700 600 500 400 300 200 150 100 1.2) 11 1000 900 800 700 600 500 400 300 200 150 100
A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100 1.2 A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100
) 11 1000 900 800 700 600 500 400 300 200 150 100 1.2) 11 1000 900 800 700 600 500 400 300 200 150 100
1.3
6 SizeMarker•©2010AppliChem
DNA Ladder 100 bp A5191
Description 100bpDNAladderisidealfordeterminingthesizeofdouble-strandedDNAfrom100to1000basepairs.The100bpand500bpfragmentsarepresentatincreasedintensitytoalloweasy
identification.Allfragmentsareblunt-ended.
Number of bands 10
Fragment sizes (bp) 1000,900,800,700,600,500x2,400,300,200,100x2
Storage buffer 10mMTris·HCl(pH7.8),10mMNaCl,1mMEDTA
Concentration 0.2mg/ml
Storage -20°C
Loading volume 1µgperlane
Package sizes A5191,0005 0.05mg(=250µl)
A5191,0025 0.25mg(=1.25ml)
Assay conditions: 1.7 % agarose
DNA Ladder 100 bp equalized (lyophilised) A3302DNAsizestandardformedium-sizedfragmentsandPCRproducts
Description Withthismarker,thebandmassofthelargerfragmentshavebeenreducedandhavebeenreinforcedforthesmallerfragments(ascomparedtomarkerA3470)byuptoafactoror4.5.Theresultisanevenappearanceinintensityoftheindividualbands. Themolnumberofthefragmentsdecreasesfromthelargertothesmallerfragments,asdothepossiblemarkingpositionsontheterminalEcoRIgeneratedrecessedends. The500bpband,withanincreaseinmassofafactorof3,enablesrapidorientationon1.5-2.0%agarosegels.Thankstotheevendistribution,ashortermigrationdistanceisneededforthelargerfragments,inordertoachieveacleardistinctionbetweenthebands. Thebestresultsareachievedafteramigrationdistanceofapprox.60-80mmonagarosegels.Eachlaneofanormalsizedagarosegel(approx.80ml)shouldbeloadedwithapprox.0.2-0.5µgofmarker.Theequalized100bpladderisthereforeveryeconomicalinuse.
Loadingbufferissuppliedseparately.
Number of bands 11
Fragment sizes (bp) 1000,900,800,700,600,500(x3),400,300,200,150,100
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;loading buffer (1X) 10%glycerol;0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.2-0.5µgperlane
Storage -20°C
Package size A3302,0020 20µg
DNA marker
ThequalityoflyophilisedDNAsizingstandardsdiffersconsiderablyfromproductsofferedbymostothermanu-
facturers thanks to the elaboratemanufacturingmethod,which also fulfils the highest quality requirements!
ThedigestedDNAisnotsimplyprecipitatedandresuspended,butalsoextractedinphenol.Thisguaranteesthe
extremelylongshelf-lifewithoutanylossofquality.Notonlythepricesshouldthereforebecompared.
©2010AppliChem•SizeMarker 7
DNA Ladder 100 bp plus A5216
Description 100bp+1.5kbDNALadderissuitableforsizinglineardouble- strandedDNAfragmentsfrom0.1to1.5kb.Useincombinationwitha loadingbuffer(pleaseseeourpresentcatalogforalistofloading buffers).
Number of bands 11
Fragment sizes (bp) 1500,1000(x2),900,800,700,600,500(x2),400,300,200,100(x2). The500bpand1kbbandsarebrighterthantheotherbandsintheladder.
Concentration Themarkerissuppliedinaconcentrationof0.2mg/ml in10mMTris·HCl(pH8.0),1mMEDTA,10mMNaCl.
Storage Storeat-20°C.Preparesmallaliquotstopreventrepeated freeze&thawing!
Loading Werecommendloadingof0.4-0.6µg(2-3µl)perlane.
Package sizes A5216,0005 0.05mg(=250µl)
A5216,0025 0.25mg(=1.25ml) Assay conditions: 1.7 % agarose
DNA Ladder Mix 100-5000 (lyophilised) A3660DNAsizestandardforthedeterminationofthesizeofmedium-sizedfragmentsandplasmids
Description The 100 bp ladder (A3470) was extended with fragments in the size range of 1500-5000. The extension by 40 % mass in the larger fragments means easy
orientationfrom1500bpupwardson1.2-1.5%agarosegels.InadditiontosmallandlargeplasmidinsertionsandPCRproducts,thesizeandquantityofplasmidvectorscan also be determined. The best results are achieved after a migration distance of 80-90 mm on agarose gels with loading volumes of 0.5-0.8 µg per lane on
standardsizedgels.
Number of bands 17
Fragment sizes (bp) 5000,4000,3000,2500,2000,1500,1000,900,800,700,600, 500(x2),400,300,200,150,100.
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;loading buffer (1X) 10%glycerol;0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.5-0.8µgperlane(forgoodstainingpropertieswithethidiumbromide)
Migration distance Bestresultsareachievedafteramigrationdistanceof70-90mm.
Storage -20°C. Avoid repeated thawing and refreezing. Portioning in small aliquots is recommended. The lyophilised product is stable for many weeks even at room temperature!
Package size A3660,0050 50µg
This sizing standard wasmanufactured using digestion of 7 plasmids withEcoR I. The plasmids are legallyprotected.Permissiontoproducecopiesmustbesoughtfromthemanufacturer.TheDNAwasdeproteinised,precipitatedandlyophilisedafterdigestionwithphenol/chloroform.Ifthefragmentsaretobelabeled,themarkershouldbedissolvedindistilledwater(10minutesatroomtemperature,shakingoccasionally).
8 SizeMarker•©2010AppliChem
DNA Ladder 250 bp (lyophilised) A3982UniversalDNAsizestandardforthedeterminationofthesizeofplasmidsandtheirDNAinsertions
Description With its twoclearly reinforcedbandsat2500and1500basepairs, the250bp ladderenables rapidorientationon1.0-1.2%agarosegels.Usinggelelectrophoresis,withintervalsof250base
pairsinarangeof250-3000basepairs, it ispossibleafterrestrictiontodetermineclearlyandprecisely,forexample,largeplasmidinsertions,andvectorplasmidsabove3000basepairswithintervalsof1-2kb.IncomparisontoDNAmarkerswithequimolarbanddistribution,themassofthesmallerbandsofthis250bpladderupto1000bphasbeenreinforcedandthelargerbandsabove3000bphavebeenreduced.Thismeans thatoptimumresultsarealreadyachievedafteramigrationdistanceof60-80mmonagarosegelsand thatsmallamountsofabout0.7µgper
lanecanbeusedwithstandardsizedagarosegels(80ml).
Number of bands 16
Fragment sizes (bp) 8000,6000,5000,4000,3000,2750,2500(x3),2250,2000,1750,1500(x3),1250,1000,750,500,250
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.4-0.8µgperlane(forgoodstainingpropertieswithethidiumbromide)
Migration distance Bestresultsareachievedafteramigrationdistanceof75-85mm.
Storage -20°C.Avoidrepeatedthawingandrefreezing.Portioninginsmallaliquotsisrecommended.Thelyophilisedproductisstableformanyweeksevenatroomtemperature!
Package size A3982,0050 50µg
DNA Ladder 250 bp plus (lyophilised) A8640DNAsizestandardforgenomicDNA,plasmidsandtheirinsertions
Description TheDNALadder250bpplusisintendedforuseinthesizingofDNAfragmentsduringcloningandofgenomicDNA.Bycombiningtwogroupsof fragmentsdifferinginthefragmentsizerange,smallerfragments(250-2000bp)aswellasplasmidvectorsandfragmentofgenomicDNA(3000-12000bp)mybesizedonthesamegel.Themassofthelargerfragmentsisreducedtoachieveabetterseparationonshortdistancesof70-90mmonly.TheterminalEcoRIrestrictionsitesallowforanefficientlabelingofthefragments.Bestresultsareachievedbyloading0.8-1.0µg/laneona1.2%agarosegel(standardminigelsize).TheDNAissupplieddeproteinizedandlyophilized.
Number of fragments 15bands
Fragments sizes (bp) 12000,10000,8000,6000,5000,4000,3000,2000,1750,1500,1250,1000,750,
500,250
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.4-0.8µg/lane(e.g.1.0-1.2%agarosegel)
Storage Storeat-20°C.Storeassmallaliquots,ifresuspended.
Package size A8640,0050 50µg
This sizing standard was manufactured using digestion of plasmids with EcoR I. The plasmids are legally protected.Permissiontoproducecopiesmustbesoughtfromthemanufacturer.TheDNAwasdeproteinised,precipitatedandlyophilisedafterdigestionwithphenol/chloroform.Ifthefragmentsaretobelabeled,themarkershouldbedissolvedindistilledwater(10minutesatroomtemperature,shakingoccasionally).
©2010AppliChem•SizeMarker 9
DNA Ladder 1 kb (lyophilised) A2667
Description Thesizeofmedium-sizedplasmidsandfragmentscanbedeterminedwith11DNAbands between10,000and500basepairs.Unliketheequimolardistributionofthebandsinother
DNAmarkers and the longmigrationdistancenecessarywith these for the separationof large fragments, the mass of the larger bands of the 1 kbp DNA ladder has beenreduced. This permits relatively short separation distances for a 1 kbp DNA ladder(approx.70mmon1.2%agarosegels) andmeans that it is very economical inuse(0.4-0.6 µg per lane, or 400 separations) on normal sized gels. Loading buffer issupplied separately. The fragments have terminal EcoR I generated recessed ends.
TheDNAisdeproteinizedandlyophilized.
Number of bands 11
Fragment sizes (bp): 10000,8000,6000,5000,4000,3000,2500,2000,1500,1000,500
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.4-0.6µgperlane
Storage -20°C
Package sizes A2667,0050 50µg
A2667,0200 4x50µg
DNA Ladder 1 kb A5207
Description 1 kbDNA Ladder is a convenientmarker for determining the size of double-strandedDNA from 250 to 10,000 base pairs. The 1,000 and
3,000bpfragmentshaveincreasedintensityrelativetotheotherbandsonethidiumbromide-stainedagarosegels,andserveasreference
indicators.Allfragmentsareblunt-ended.
Number of bands 13
Fragment sizes (kb) 10.0,8.0,6.0,5.0,4.0,3.0(x2),2.5,2.0,1.5,1.0(x2),0.75, 0.5,0.25
Storage buffer 10mMTris·HCl(pH7.8),10mMNaCl,1mMEDTA
Concentration 0.2mg/ml
Storage -20°C
Package sizes A5207,0005 0.05mg(=250µl)
A5207,0025 0.25mg(=1.25ml)
DNA marker
10 SizeMarker•©2010AppliChem
DNA Ladder 1 kb concatamer (lyophilised) A6430DNAsizemarkerasalternativetoirregularl-fragments(from1toapprox.25kb)Description Apartial ligationof1000bpHind III fragments results inaDNA ladder ranging from1000 to approx. 22000 bp in 0.8 to 1% gels using standard agarose (Prod.-No. A2114; separation
range up to approx. 25 kbp). The upper limit of the fragment ligation results in the mainmass of the ligation products in the separation range optimal for standard agaroses (agar). Forabetterorientation,the3000bpand10,000bpbandsarebrighter.Optimalresultswillbeachievedafteradistanceofapproximately80-100mminagarosegelsandaloadingvolumeofapproximately1µgperlaneinnormal-sizedgels.This1000bpconcatamerladdermaybeappliedeitherinreducedloadingvolumes(foraseperationrangee.g.<10000bp)orinincreasedvolumes(>15000bp). Evenunderoptimizedligationconditions,anintramolecularreligationmayoccur,especiallywithsmallerfragments.Therefore,athighgelloadingvolumes,ligationproducts(closedcircles)havebeen
observedbetweenthe1000and3000bpbands.Forabetterorientation,the3000bpbandisbrighter.
Number of bands >25Fragment sizes (bp) 1000bpsteps(1000-approx.25000)
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume approx.1.0µgperlane
Migration distance Optimumresultsafteradistanceof15-25mmonly.Storage -20°C.
Package size A6430,0050 50µg
DNA Marker quick-run (lyophilised) A7215DNAsizestandardfordeterminationofDNAfragmentsaftershortgelruns(15-25mm)
Description Thismarkerisidealforusewithminigels,sinceitsbandsareseparatedverywellevenafterveryshortgelrunsasshortas15-25mminane.g.1.5-1.8%agarosegel.Thesinglebandshavebeenequalizedintermsoftheirmass.The1500bpfragmenthasanincreasedmassforbetterorientation.Optimumresultsareachievedina1.5-1.8%agarosegelwithaslittleas0.25µgofthemarkerperlane(normalsizedminigel).Pleasenote that theDNA fragments have to be saturated with ethidium bromide in
therunningbufferduringtheseparation.
Number of bands 5
Fragment sizes (bp) 2500,2000,1500*,1000,500 Themassofthemarkedfragment*hasbeenincreasedforbetterorientation.
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.015%bromophenolblue
Loading volume 0.25µgperlane(foragooddetectionwithethidiumbromide)
Migration distance Optimumresultsafteradistanceof15-25mmonly.
Storage -20°C.Preventrepeatedfreezingandthawing(>20x).Werecommendtoprepare smallaliquots.Thelyophilisedformisstableevenforweeksatambienttemperature
Package size A7215,0050 50µg
ThismarkerhasbeendesignedasanalternativeforirregularλDNAfragmentmarkers,fortheseparationinstandardlowendoosmosisagarose(Prod.-No.A2114;separationrange70bptoapproximately25kbp).Incomparisontootherconcatamermarkers,ithasareducedmassinthenon-separatingrange(highmolecularweightrange),resultinginabetterresolution(goodreadingsupto24kbp).Forapplicationsrequiringagoodseparationinthehighmolecularweightrangepulsed-fieldelectrophoresisisrecommended.Duetotheproductionprocedure(ligationof1000bpHindIIIfragments),itisimpossibletodeterminethespecificmassofasingleband.TheDNAofthismarker(50µgsufficientforapproximately100loadings)isdeproteinised,Iyophilisedandissuppliedwith1mlofthe1xgelloadingbuffer.
Thismarker ismadeofplasmidswithspecificmutagenesissites.TheEcoRI-digestedDNAhasbeendeproteinizedwithphenol/chloroform,desalted,precipitatedandlyophilised.ResuspendtheDNAinthesterile-filteredgelloadingbuffer(suppliedwiththemarker)byincubationfor10minutesatroomtemperaturewithoccasionalshaking.
©2010AppliChem•SizeMarker 11
DNA Marker quick-run extended (lyophilised) A7222DNAsizestandardfordeterminationofDNAfragmentsaftershortgelruns(35mm)
Description Thismarker is ideal for usewithmini ormidi gels, since its bands are separatedverywellevenafterveryshortgelrunsasshortas35mminane.g.1.2%agarosegel. The single bands have been equalized in terms of their mass. The 1500 bpfragmenthasanincreasedmassforbetterorientation.Optimumresultsareachieved
withaslittleas0.3-0.5µgofthemarkerperlane(normal-sizedmini/midigel).
Number of bands 9
Fragment sizes (bp) 6000,4000,2500,2000,1500*,1000,500,200,100 Themassofthemarkedfragment*hasbeenincreasedforbetterorientation.
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.015%bromophenolblue
Loading volume 0.3-0.5µgperlane (foragooddetectionwithethidiumbromideinaminiormidigel)
Storage -20°C.Prevent repeated freezingand thawing(>20x).Werecommend toprepare
smallaliquots.Thelyophilisedformisstableevenforweeksatambienttemperature!
Package size A7222,0050 50µg
Thismarkerismadeofplasmidswithspecificmutagenesissites.TheEcoRI-digestedDNAhasbeendeproteini-zedwithphenol/chloroform,desalted,precipitatedandlyophilised.ResuspendtheDNAinthesterile-filteredgelloadingbuffer(suppliedwiththemarker)byincubationfor10minutesatroomtemperaturewithoccasionalshaking.
DNA Marker pBR322 - Hae III (lyophilised) A4406DNAsizestandardforhigh-percentageagarosegelsandpolyacrylamidegels
Description Plasmid pBR322 was digested with Hae III, deproteinized and lyophilized. This marker is particularly suited to comparisons with small PCR fragments and DNA
from restriction samples on polyacrylamide gels or high-percentage agarose gels (> 2.2 %). The best results are achieved after a migration distance of approx. 70-80mmonagarosegelsor100mmon6-8%polyacrylamidegels.Eachlaneofanormalsizedgel(approx.80ml)shouldbeloadedwithapprox.1µgof marker
foragarosegelsor0.5µgforPAAgels.Loadingbufferissuppliedseparately.
Number of bands 22
Fragment sizes (bp) 587,540,502,458,434,267,234,213,192,184,124,123,104,89,80,64,57, 51,21,18,11,8
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.5-1.0µgperlane
Storage -20°C
Package size A4406,0050 50µgPlease note! Extremelysmallfragmentscanonlybevisualizedonhigh-percentagePAAgels.
DNA marker
12 SizeMarker•©2010AppliChem
DNA Marker pBR328 Mix (lyophilised) A6927DNAsizestandardformiddle-sizedfragmentsandPCRproductsDescription PlasmidpBR328wasdigestedwithHinfIandBglI,respectively,deproteinisedand
lyophilised. The fragments have beenmixed in an equimolar ratio. Thismarker isparticularlysuitableforcomparisonwithPCRfragments(e.g.fooddiagnostics)andDNAfromrestrictionsamplesonagarosegelswithsizesbetween150and2000basepairs.Theconcisedistributionoffragmentsofthemarkermakesthisproductidealfor long and short gel runs with running distances of 70-80mm (1.5% agarosegel).Load1µgofthemarkerperlaneofanormalsizedagarosegel(approx.80ml
Loadingbufferissuppliedseparately.
Number of bands 12
Fragment sizes (bp) 2176;1766;1230;1033;653;517;453;394;298;234;220;154
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 1.0µgperlane
Storage -20°C.Prevent repeated freezingand thawing(>20x).Werecommend toprepare smallaliquots.Thelyophilisedformisstableevenforweeksatambienttemperature!
Package sizes A6927,0050 50µg
ResuspendtheDNAinthesterile-filteredgelloadingbuffer(suppliedwiththemarker)for10minutesatroom
temperaturebyoccasionalshaking.
DNA Marker pBR322 - Hae III A5229
Description ThismarkerisgeneratedbydigestionoftheplasmidpBR322withHaeIII.
Number of bands 22
Fragment sizes (bp) 587,540,502,458,434,267,234,213,192,184,124,123,104,89,80,64,57, 51,21,18,11,8
Storage buffer 10mMTris·HCl(pH7.8),10mMNaCl,1mMEDTA
Concentration 0.2mg/ml
Storage -20°C
Package sizes A5229,0005 0.05mg(=250µL)
A5229,0025 0.25mg(=1.25ml)
Assay conditions: 1.7 % agarose
DNA marker
©2010AppliChem•SizeMarker 13
DNA Marker Phage Lambda - Sty I A5194
Description Lambda DNA (cI857 Sam 7) Sty I markers are prepared by digesting Lambda DNA with Sty I, followed by inactivation of the enzyme. The DNA fragments arethenethanol-precipitatedandresuspendedinthestoragebuffer.
Number of bands 11
Fragment sizes (bp) 19329*;7743;6223;4254*;3472;2690;1882;1489;925;421;74
Storage buffer 10mMTris·HCl(pH8.0),1mMEDTA
Concentration 0.2-0.5µg/µl
Storage -20°C
Package sizes A5194,0005 0.05mg(=250µl)
A5194,0025 0.25mg(=1.25ml)Please note! Thecohesiveendsoffragments1and4(*)maycausetheformationofanextraband(23583bp). Thefragmentsmaybeseparatedbyheatingto65°Cfor3minutesbeforeloadingthesampleontothegel.
DNA Marker Phage Lambda - BstE II (lyophilised) A4412DNAsizestandardmadeofphagelDNAforthedeterminationoflargeDNAfragments(digestionofgenomicDNA)Description Natural lambdaDNAwasdigestedwithBstE II, deproteinizedand lyophilized.This markerissuitableforcomparisonswithfragmentsizesbetween8400and700base
pairs.Thebestresultsareachievedafteramigrationdistanceofapprox.80-90mmon1.0-1.2%agarosegels.Eachlaneofanormalsizedgel(approx.80ml)should
beloadedwithapprox.1µgofmarker.Loadingbufferissuppliedseparately.
Number of bands 14
Fragment sizes (bp) 8454*,7242,6369,5686*,4822,4324,3675,2323,1929,1371,1264,702, 224,117
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 1.0µgperlane
Storage -20°C
Package size A4412,0100 2x50µgPlease note!Thecohesiveendsoffragments1and4(*)mayformanadditionalband(14140bp).Beforeloadingontothegel,thefragmentscanbeseparatedbyheatingto65°Cfor5minuteswithsubsequentincubationonice.
DNA marker
14 SizeMarker•©2010AppliChem
DNA Marker Phage Lambda - BstE II A5220
Description Phage Lambda (cI857 Sam 7)DNA/BstE II markers are prepared by digesting Lambda DNA with BstE II, followed by inactivation of the enzyme. The DNA fragmentsarethenethanol-precipitatedandresuspendedinthestoragebuffer.
Number of fragments 14
Fragment sizes (bp) 8454*,7242,6369,5686*,4822,4324,3675,2323,1929,1371,1264,702, 224,117
Storage buffer 10mMTris·HCl(pH8.0),1mMEDTA
Concentration 0.2-0.5µg/µl
Storage Storeat-20°C
Package sizes A5220,0005 0.05mg(=250µl)
A5220,0025 0.25mg(=1.25ml)Please note!Thecohesiveendsoffragments1and4(*)maycausetheformationofanextraband(14140bp). Thefragmentsmaybeseparatedbyheatingto65°Cfor3minutesbeforeloadingthesampleontothegel.
DNA Marker Phage Lambda - Hind III (lyophilised) A5589DNAsizestandardmadeofphagelDNAforthedeterminationoflargeDNAfragments(digestionofgenomicDNA)Description NaturallambdaDNAwasdigestedwithHindIII,deproteinizedandlyophilized.This marker is suitable for comparisonswith large tomedium-sized fragments between
23,000and600basepairs.Thebestresultsareachievedafteramigrationdistanceofapprox.80-90mmon1.0-1.2%agarosegels.Eachlaneofanormalsizedgel(approx.80 ml) should be loaded with approx. 0.5-0.8 µg of marker. Loading buffer is
suppliedseparately.
Number of bands 8
Fragment sizes (bp) 23130*;9416;6557;4361*;2322;2027;564;125
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.5-0.8µgperlane
Storage -20°C
Package size A5589,0100 2x50µgPlease note! Thecohesiveendsoffragments1and4(*)mayformanadditionalband(27491bp).Beforeloadingontothegel,thefragmentscanbeseparatedbyheatingto65°Cfor5minuteswithsubsequentincubationonice.
DNA marker
©2010AppliChem•SizeMarker 15
DNA Marker Phage Lambda - Hind III A5223
Description LambdaDNA(cI857Sam7)/HindIIImarkersarepreparedbydigestingLambdaDNAwithHind III, followedbyheat-inactivationof theenzyme.TheDNAfragmentsare thenethanol-precipitatedandresuspendedinstoragebuffer.
Number of bands 8
Fragment sizes (bp) 23130*;9400;6557;4361*;2322;2027;564;125
Storage buffer 10mMTris·HCl(pH8.0),1mMEDTA
Concentration 0.2-0.5µg/µl
Storage -20°C
Package sizes A5223,0005 0.05mg(=250µl)
A5223,0025 0.25mg(=1.25ml)Please note! Thecohesiveendsoffragments1and4(*)maycauseformationofanextraband(27491bp). Thefragmentsmaybeseparatedbyheatingto65°Cfor3minutesbeforeloadingthesampleontothegel.
DNA Marker pUC19 - Msp I A5235
Description DigestionofthepUC19plasmidyields12fragments.
Number of bands 12(9visible)
Fragment sizes (bp) 501,489,404,331,242,190,147,111,110,67,34x2,26
Storage buffer 10mMTris·HCl(pH7.8),10mMNaCl,1mMEDTA
Concentration 0.2mg/ml
Storage -20°C
Package sizes A5235,0005 0.05mg(=250µl)
A5235,0025 0.25mg(=1.25ml)
Assay conditions: 1.7 % agarose
DNA Marker pUC19 - Msp I (lyophilised) A3996DNAsizestandardforhigh-percentageagarosegelsandpolyacrylamidegels
Description PlasmidpUC19wasdigestedwithMspI,deproteinizedandlyophilized.Thismarkerisparticu-larlysuitableforcomparisonswithsmallPCRfragmentsandDNAfromrestrictionsamplesonpolyacrylamidegelsorhigh-percentageagarosegels.Thebandsat501/489bpand111/110bphaveapprox.doublemassandproviderapidorientationafterashortmigrationdistanceon2.0-2.2%agarosegels.Thebestresultsareachievedafteramigrationdistanceofapprox.50-70mmonagarosegelsor100mmon6-8%polyacrylamidegels.Eachlaneofanormalsizedagarosegel (approx.80ml) shouldbe loadedwith approx.0.5-1.0µgofmarker. Loadingbuffer issuppliedseparately.
Number of bands 12
Fragment sizes (bp) 501,489,404,331,242,190,147,111,110,67,34,26
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Loading volume 0.5-1.0µgperlane
Storage -20°C
Package size A3996,0050 50µgPlease note! Extremelysmallfragmentscanonlybevisualizedusinghigh-percentageandhigh-qualityagarosegels(>2.2%)withlargemassoffragments.
16 SizeMarker•©2010AppliChem
dyes for nucleic acidsProd.-No. Description DNA/RNA Dye Complex Excitation/Emission Complex Excitation/Emission Complex (Detection)
dyes for nucleic acids(Detection)
dyes for nucleic acids Sensitivity recommended working
A1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°CA1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°CA1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°C ssDNA/RNAredfluorescence
A0691 Crystalviolet DNA(purple-red) 590nm 10ng 1µg/ml
A1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwaterA1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwaterA1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwater intercalationintoGC-Basepairs;white-bluefluorescence
A1151 Ethidiumbromide* DNAintercalation 260-360nmor546nm/590nm 0.5ng 0.2-0.5µg/ml 10mg/mlinwater;2-8°C
A2388 Malachitegreenoxalate DNA 626nm A2388 Malachitegreenoxalate DNA 626nm
A1402 Methyleneblue DNA,RNA-stainingatacidicpH 297nm/672nm 40ng 0.2%in0.4MNaOAc/0.4MAceticacid
A5595 DNA-DyeMethyleneblue DNA 297nm/672nm 40-80ng 200-foldconcentratedsolution;2-8°CA5595 DNA-DyeMethyleneblue DNA 297nm/672nm 40-80ng 200-foldconcentratedsolution;2-8°C
A1403 Methylgreen specificbindingtoAT-richsequences;DNA(green) 638nm 1µg/ml
A0581 Methylorange(C.I.13025) 10ng 0.0005% 0.25%inwaterA0581 Methylorange(C.I.13025) 10ng 0.0005% 0.25%inwater
A3918 Nileblue DNAintercalation(blue) 630nm/673nm 40ngonagarose;4ngondriedgel 1µg/ml 1µg/mlin0.25XTAEbuffer(pH8.0)
A2261 Propidiumiodide DNA-intercalator;nouptakeintolivingcells 530nm/625nm 1µg/ml 2-8°CorRTA2261 Propidiumiodide DNA-intercalator;nouptakeintolivingcells 530nm/625nm 1µg/ml 2-8°CorRT
A1406 PyroninY DNA/RNA(red) 488-530nm/565-574nm 0.05%or1µg/ml
A3944 Silvernitrate DNA(brown) 2.5ngdsDNAonagarose A3944 Silvernitrate DNA(brown) 2.5ngdsDNAonagarose
A1400 Stainsall RNA(blue-violet)
*Thereareseveralready-to-usesolutionsavailable! A1152Ethidiumbromide-Solution1%BioChemica DAPI(4',6-Diamidino-2-phenylindoledihydrochloride);PBS(Phosphate-bufferedsaline);NaOAc(Sodiumacetate)A2273Ethidiumbromide-Solution0.07%“dropper-bottle” PBS(Phosphate-bufferedsaline)
Protocol for staining with DNA-Dye Methylene blue:Approx.50ml1XDNA-DyeMethylenebluestainingsolutionarerequiredtostainanagarosegelwiththedimensionsofapprox.80x60mm(widthxlength)andathicknessof3-4mm.The gel should be trimmed down appropriately, with the edge of a ruler for example, toremoveareasofthegelthatwerenotusedforDNAseparation.Thestainingisperformedinanadequatelysizeddish,inwhichthegelisfloatedtoadepthofabout5mmwithdyesolution.To prepare theDNA-DyeMethylene blue staining solution for use, 250µl are dissolved in50mlwater.Thegelislefttostainforabout20minutesandthedishisshakenoccasionally.Torevealthebands(differentstainingintensities)thegelisdestainedwithtapwaterseveral
timesforabout5-10minutes.Theblue-stainedDNAbandsareclearlyvisibleintransmittedlightandcanbemeasuredwitharulerordocumentedphotographically.MethylenebluedoesnotstainDNApermanently:dependingonthethicknessofthegelandthestoragetempe-rature,theDNAbands(especiallysmallerfragments)maylosetheirstainingaftersometime.
DNA-Dye Methylene blue A5595Nontoxicsolutionforthestainingofnucleicacidsinagarosegels
Amount 10ml200-foldmethylenebluedyeconcentrate
Storage 2-8°C
Stability 18-24months(shakewellbeforeuse)
Package size A5595,0010 10ml
The DNA marker Phage Lambda-Hind III (A5589) was loaded on the 1st lane (*).
2.1
2.2
*
©2010AppliChem•SizeMarker 17
dyes for nucleic acids Sensitivity recommended working concentration Stock solution / Storage
A1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°CA1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°C
A0691 Crystalviolet DNA(purple-red) 590nm 10ng 1µg/ml
A1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwaterA1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwater
0.5ng 0.2-0.5µg/ml 10mg/mlinwater;2-8°C
A2388 Malachitegreenoxalate DNA 626nm
A1402 Methyleneblue DNA,RNA-stainingatacidicpH 297nm/672nm 40ng 0.2%in0.4MNaOAc/0.4MAceticacid
A5595 DNA-DyeMethyleneblue DNA 297nm/672nm 40-80ng 200-foldconcentratedsolution;2-8°C
A1403 Methylgreen specificbindingtoAT-richsequences;DNA(green) 638nm 1µg/ml
A0581 Methylorange(C.I.13025) 10ng 0.0005% 0.25%inwater
A3918 Nileblue DNAintercalation(blue) 630nm/673nm 40ngonagarose;4ngondriedgel 1µg/ml 1µg/mlin0.25XTAEbuffer(pH8.0)
A2261 Propidiumiodide DNA-intercalator;nouptakeintolivingcells 530nm/625nm 1µg/ml 2-8°CorRT
A1406 PyroninY DNA/RNA(red) 488-530nm/565-574nm 0.05%or1µg/ml
A3944 Silvernitrate DNA(brown) 2.5ngdsDNAonagarose
Abbreviations DAPI(4',6-Diamidino-2-phenylindoledihydrochloride);PBS(Phosphate-bufferedsaline);NaOAc(Sodiumacetate)
A2273Ethidiumbromide-Solution0.07%“dropper-bottle” PBS(Phosphate-bufferedsaline)
Repeated use of the 1X staining solutionThe prepared 1X methylene blue staining solution can be kept in a suitable brown-glass bottle at 2-8°C for4-6monthsandcanbeusedseveraltimes.Thestainingperiodshouldbelengthenedbyabout20%fortheseconduse,andbythesameagainforthethirduse.Afterthis,thestainingcapacityofthesolutionislargelyexhaustedandanewsolutionshouldbeprepared.
SensitivityThebandsina1kbladder(loadingamount1µg)canbestainedwithnoproblems.Smallfragments(150bp)canalsobestained.Thedecisivefactorsinsensitivityarethethicknessofthegel,thegel:stainingsolutionratio,andthedestainingtime.Byvaryingthestainingconditions,youcaneasilydeterminethebestconditionsforyoursystem.
HandlingAlaboratorycoatandglovesshouldbewornwhenhandlingthestainingsolution.
PrecautionOncontactwiththeskinorclothing,theareashouldbewashedwithlargeamountsofsoapandwater.
Short protocol1. DiluteDNA-DyeMethyleneblue200Xconcentratedownto1X(250µlDNA-DyeMethyleneblue+50mlwater)2.Trimdowntheagarosegeltothesmallestareapossible.3. Incubate the gel (dimensions approx. 80 x 60 mm) with 50 ml DNA-Dye Methylene blue for about 20 minutes;shakeoccasionally.Thestainingsolutionshouldcoverthegeltoadepthofabout5mm.
Please note:thestainingsolutioncanbeusedseveraltimes!4.Destainrepeatedlyinwaterfor5-10minutes.
18 SizeMarker•©2010AppliChem
Ethidium bromideisthemostcommonlyuseddyeforstainingDNAduringorafterpolycrylamideoragarosegelelectrophoresis.Excitationwithlightatawavelenghtof254nmisabsorbedbytheDNAandtransferredtoethidiumbromide.Excitationat366nmdirectlyexcitesthedye(3).Anaqueousstocksolutionispreparedataconcentrationof10mg/mlandemployedat0.2-0.5µg/ml(5).Stainingisperformedafterthegelrun(especiallyPAGE)orduringthegelrun(agarose).ThelatterallowstheobservationofthegelrunbycontrolunderUVlight.Ethidiumbromideisaddedtotheagarosesolution.Pleasenote,that'prestaining'mayleadtoartifacts(4).In another application, ethidium bromide may be used to sensitively quantify DNA by spectrofluorimetry.Excitationat250nmwithUVlightandanemissionat605nm,thesensitivityisevenbetterascomparedtothedyeHoechst33258.Ataconcentrationof0.5µg/mlEtBr,thedetectionlimitof10ng/mlDNAisreachedwithalinearrangeofmeasurementfrom20to1250ng/ml(6).Caution:Ethidiumbromideisapowerfulmutagenandismoderatelytoxic.Glovesshouldbewornwhenworkingwithsolutionsthatcontainthisdye,andamaskshouldbewornwhenweighingethidiumbromide.
dyes
for
nucl
eic
acid
s
Ethidium bromide - Solution 1 % BioChemica A1152Synonym 2,7-Diamino-10-ethyl-9-phenylphenanthridiumbromide-Solution
Composition Ethidiumbromide:10mg/ml filteredsolution
Storage Storethestocksolutionat2-8°Cprotectedfromlight.
Package sizes A1152,0025 25ml
A1152,0100 100ml
Ethidium bromide - Solution 0.07 % “dropper-bottle” A2273Synonym 2,7-Diamino-10-ethyl-9-phenylphenanthridiumbromide-Solution
Composition Ethidiumbromide:0.7mg/ml filteredsolution
Storage Storethestocksolutionat2-8°Cprotectedfromlight.
Description EthidiumbromideactsasaDNAintercalatorandhasaconsiderablemutagenicpotential.InordertoreducethepossiblecontacttoethidiumbromideAppliChemprovidesaready-to-usesolutionina'dropperbottle'.Theconcentrationofethidiumbromidesolution(0.7mg/ml)isadjustedsothattheadditionofonedrop(ofavolumeof50µl)ofthissolutionissufficienttostain50mlagarosegelsolution.
Package sizes A2273,0005 5ml A2273,0015 15ml
Literature(1) Waring, M. (1975) in Antibiotics Vol. III , 141-165 (J.W. Corcoran & F.E. Hahn eds.) Springer-Verlag: Review article about ethidium and propidium.(2) Lunn, G. & Sansone, E.B. (1987) Anal. Biochem. 162, 453-458 Ethidium bromide: Destruction and decontamination of solutions.(3) Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring
Harbor, New York.(4) Gärtner, U. et al. (1996) Anal. Biochem. 243, 194-196 Apparent degradation of cleaved genomic DNA may be an ethidium bromide prestaining artifact.(5) Lucey, M.J. et al. (1997) BioTechniques 23, 780-782 Reducing the ethidium bromide quantity in agarose gel electrophoresis.(6) Bonasera, V. et al. (2007) BioTechniques 43, 173-176 Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using EtBr.
2.3
©2010AppliChem•SizeMarker 19
Protein Marker I (14-116) A5238Description Thebandsof3µlmarkerareeasily visualizedwithCoomassiestainingina
polyacrylamidegel.ProteinMarkerIisamixtureof7purifiedproteinssuppliedingelloadingbufferfordirectapplicationto
anSDSpolyacrylamidegel.
Number of bands 7
Fragment sizes (kDa) 116.0;97.4;66.2;37.6;28.5;18.4;14.0
Loading buffer 50mMTris·HCl(pH6.8), 100mMdithiothreitol,2%SDS, 0.1%bromophenolblue,10%glycerol
Assay conditions 3µl/12%PAGE
Package size A5235,0500 500µl
protein marker
precisionprecision
Prod. Protein Marker Bands Fragment Sizes [kDa] No.
A5238 ProteinMarkerI(14-116) 7 116 97.4 66.2 37.6 28.5 18.4 14
A5418 ProteinMarkerII(6.5-200)prestained 8 200 116 68 43 30 20 14.4 6.5
A4402 ProteinMarkerIII(6.5-200) 8 200 116 68 43 30 20 14.4 6.5
A3993 ProteinMarkerIV(10-150) 8 150 100 80 60 40 30 20 10
A8359 ProteinMarkerV(10-175)prestained 11 175 130 95 70 62 51 42 29 22 1410.5
A8889 ProteinMarkerVI(10-245)prestained 12 245 180 135 100 75 63 48 35 25 20 17 11
General ConsiderationsSizeestimation:Prestainedproteinmarkersarenotrecommendedforprecisedeterminationofthemolecularweightsizesincetheirbehaviorduringelectrophoresisstronglydependsonelectrophoresisconditions.Forexactdeterminationsofproteinsizesuseunlabeledmarkerproteins,i.e.non-prestainedmarkers.
Proteinblotting:Thetransferofproteinsduringblottingdependsontheirsize(e.g.lysozymeisfullytransfer-redafter30minutes,whilemyosinrequires2.5hours@1V/cm2).
3.13.13.13.13.1
3.2
20 SizeMarker•©2010AppliChem
Protein Marker IV (10-150) A3993Recombinantproteinsizemarkerforgelelectrophoresis
Description The bands of 2.5 µl marker are easily visualized with Coomassie staining in a polyacrylamidegel.
Number of bands 8
Protein sizes (kDa) 150;100;80;60;40;30;20;10
Assay conditions 10µl/4-20%SDS-PAGEgradientgel (Tris-Glycine);Coomassiestaining
Storage -20°C, if stored longer than 1 month. Pleasenotethatrepeatedfreezingand thawingwillreduceproductquality.
Preparationofsmallaliquotsisrecommended.Thawedaliquotsarestable
foroneweekat+4°C.
Package size A3993,0500 500µl
Thisisa ready-to-usemarkercontainingrecombinantproteins.Theproductmaycontainresidualiodoacetamide.
Protein Marker III (6.5-200) A4402Description Thebandsofthe2.5µlmarkerareeasily visualizedwithCoomassiestainingina polyacrylamidegel.
Number of bands 8
Protein sizes (kDa) 200.0;116.0;68.0;43.0;30.0;20.0; 14.4;6.5
Assay conditions 5µl/4-20%SDS-PAGEgradientgel; Coomassiestaining
Storage -20°C,ifstoredlongerthan1month. Pleasenotethatrepeatedfreezingandthawingwillreducepro-
ductquality.Preparationofsmallaliquotsisrecommended.
Package size A4402,0001 1.0ml
Thisisaready-to-usemarkercontainingacylatedproteins.Theproductmaycontainresidualiodoacetamide.
precision
Application ●Theproteinemarkershavetobe“preheated”toroomtemperature,toguaranteethatallcom-ponentsaredissolved.●Apply1-5µlofthemarkertoamini-gel(10x10cm,1-0.75mmthick,7mmslot).Use1XLaemmlibuffertodilutethismarker,ifnecessary.●Tominimizemyosinaggregation,thealiquottobeloadedontothegelmaybeheatedto95°Cfor1-2minutes.
©2010AppliChem•SizeMarker 21
Protein Marker II (6.5-200) prestained A5418Prestainedproteinsizemarkerforgelelectrophoresis
Description This is a ready-to-use marker containing covalent prestained proteins. The product containsformamide.
Number of bands 8
Protein sizes (kDa) 200.0;116.0;68.0;43.0;30.0;20.0; 14.4;6.5
Assay conditions 10µl/4-20%PAGEgradientgel Tris-Glycine;leftlaneprestainedbutnot
Coomassie-stained;rightlaneprestained andadditionalCoomassiestaining.
Storage -20°C,ifstoredlongerthan1month. Pleasenotethatrepeatedfreezingand
thawingwillreduceproductquality.Pre- parationofsmallaliquotsisrecommended.
Package size A5418,0250 250µl
Protein Marker V (10-175) prestained A8359PrestainedproteinmarkerforgelelectrophoresisandWesternblotting.MagentaMarker
Description Ready-to-useprestainedproteinsizemarker.Suppliedinloadingbuffer.
Number of bands 11
Protein sizes (kDa) 175;130;95;70;62;51;42;29;22;14;10.5
Threereferenceproteinscoupledwithabluedyeforeasyidentification: approx.10,40,and90kDa.
Storage At2-8°Cformax.3months;at-20°Cforlongtermstorage.
Package size A8539,0250 250µl
prestained
3.33.3
22 SizeMarker•©2010AppliChem
Protein Marker VI (10-245) prestained A8889PrestainedproteinmarkerforgelelectrophoresisandWesternblotting.Blue-Green-RedProteinMarker
Description ProteinMarkerVIprestainedisathree-colorproteinstandardwith12prestainedproteinscoveringawidemolecularweightrangefromapprox.10to245kDa.Proteinsarecovalentlycoupledwitheitherabluechromophoreoronegreen(25kDa)andonereddye(75kDa),respectively.GelrunmaybemonitoredduringproteinseparationonSDS-PAGE(Tris-glycinebuffer,“Laemmlisystem”).The main applications are the monitoring of protein migration duringSDS-polyacrylamide gel electrophoresis, the verification of Western transferefficiency onmembranes (PVDF, nylon, or nitrocellulose), and the sizing ofproteins on SDS-polyacrylamide gels and Western blots. The size marker is
suppliedready-to-useingelloadingbuffer.
Number of bands 12
Protein sizes (kDa) 245;180;135;100;75;63;48;35;25;20;17;11
Package size A8889,0500 500µl
RecommendationsforLoading1.Thawtheladdereitheratroomtemperatureorat37-40°Cforafewminutestodissolvepre-cipitatedsolids.Donotboil!
2.Mixthoroughlytoensurethesolutionishomogeneous.3.LoadthefollowingvolumesoftheladderonSDS-polyacrylamidegel:5µlperwellformini-gels,2.5µlperwellforblots;10µlperwellforlargegels,5µlperwellforblots.
prestained3.33.3
SizeMarker•©2010AppliChem
Sometimes, less is more!
For The Sake OfThe Environment
Manyreagentspreparedinbiomedicalresearchlabsareidealnutrientbrothsforunwantedgerms(bacteria,fungi).Topreventtheirgrowth,reagentsareeitherautoclaved,sterilefiltered,orantibiotics/antimycoticsortoxicsubstancesareadded.OneoftheseadditivesisThimerosal,amercury-containingmoleculewhichisdangerousfortheenvironment.Ouroriginalimmunoasssaybuffercontainedthischemicaltoo,butnowwehavereplaceditbythenontoxicProClin® 300.Forthesakeoftheenvironment.
For Your Safety
Wewouldliketokeepyouhealthysothatyoustayourcustomer.FYI:Thimerosalisclassifiedastoxic.Thelethaldose(rat,s.c.)is9mg/kg,comparedtoethidiumbromidewithalethaldose(mouse,s.c.)of110mg/kg.Insomecountries,Thimerosalisaforbiddenadditive.
For Better Results
Changingthecompositionhasnonegativeinfluenceontheperformanceoftheproducts.WithCrossDownandallotherimmunoassaybuffersyouareeveninabettermoodandyourimmunoassaysshowabetterquality.
©2010AppliChem•SizeMarker 23
Abbreviations:BSA(BovineSerumAlbumin);CBB(Coomassie®Brilliantblue);MeOH(Methanol);RuBPS(Ruthenium(II)tris(bathophenanthrolinedisulfonate));TCA(Trichloroaceticacid);&onlyincombinationwithCoomassie®!$detectionofphosphoproteins;formationofinsolublephosphatecomplexes(sensitivity:0.1nM)
24 SizeMarker•©2010AppliChem
dyes
for p
rote
in g
els Prod.-No. Description Comment Absorption maxima Sensitivity Recommended Concentration Stock solution
A2176 BismarckbrownR increasessensitivityofCoomassie® BrilliantBlueR-250staining
lmax.468nm 25ng& CoomassieBrilliantblueR-250(0.2%w/v)BismarckbrownR(0.05%w/v)
SolventMeOH:Aceticacid:Water(40:7:53);dissolvedyesseparately,thenmix1:0.75(v/v)
A3480 Coomassie®BrilliantBlueG-250 stainsampholytesinIEFgelstoo!* <0.5ng Mix80mlofthestocksolutionwith20mlofMeOHjustbeforeuse.
0.1%w/vCBBin2%w/vPhosphoricaicd,10%w/vAmmoniumsulfate(Donotfilter!)
A1092 Coomassie®BrilliantBlueR-250 stainsampholytesinIEFgelstoo!* lmax.withprotein549nm,w/oprotein555nm
10ng 0.1%in20%MeOH,10%Aceticacid
A0822 EosinY reversiblestaining similartoCBBR-250 0.25%(w/v)in0.1NNaOH preparestainingsolutionfreshdaily;maybeusedforseveralgels;
A1346 EriochromeblackT 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid;incombinationwithRhodamineB
A1401 FastGreenFCF dyecomplexinacidicpHrange lmax.(pH8.3)615;(MeOH)620nmlmax.(withprotein)635nm
400ng 0.25%(w/v)in10%Aceticacidupto1%(w/v)in7%Aceticacid
A2385 Kongored stainingintheacidicpHrange lesssensitivethanCBB 0.1%(w/v)in0.2MAcetatebuffer(pH3.5) 1%(w/v)indistilledwaterA2388 Malachitegreenoxalate Phosphatasestainingingels Stainingsolution=
3Vol.Solution1+1Vol.Solution2(stablefor3weeks;filterpriortoeachuse)
Solution1:0.15gMalachitegreenoxalatein300mlWater;Solution2:4.2gAmmoniummolybdatetetrahydratein100ml5NHCl;
Nilered 5ngA1405 PonceauS acidicdiazodye lmax.(H2O)517-823nm 500ng 0.1-0.5%in3%TCAor1mlAceticacid
glacialad100mlWaterpreparefreshpriortouse
A7808 Proteo-DyeRuBPS lmax.617nm 2-5ng 1µM 1mMA3930 RhodamineB$ 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid;incombinationwith
EriochromeblackTA3972 Silvernitrate brown 2-5ngA1400 Stainsall** stainsdifferenttypesofproteins
indifferentcolorslmax.forBSA515nm likeCBB 0.005% 0.1%inFormamideor5.6mg/10mlin50%Dioxane;
preparefresh
Zinc-Imidazole reversiblestaining 10-20ngSDS-PAGE40-80ngnativePAGE
Coomassie® Brilliant blue R-250 (C.I. 42660) A1092Synonym BrilliantblueR,Xylenebrilliantcyanine
Order No. Quantity Formula M CAS-No.
A1092,0010 10g C45H44N3NaO7S2 825.98g/mol 6104-59-2
A1092,0025 25g
A1092,0100 100g
®registeredtrademarkofImperialIndustriesPLC
Coomassie®BrilliantBlueR-250isoneof themostcommonlyusedstains forproteins,after theirseparationbypolyacrylamidegelelectro-phoresis. The protein-dye complex has an absorptionmaximum at 549 nm, the dyewithout protein at 555 nm (in 0.01M citrate buffer,pH3).The intensity in stainingofproteinsprobablydependson thebasicityofaprotein.Perpositivelychargedaminoacidapproximately1.5-3moleculesofCoomassie®willbebound.Thisvariationcomplicatetheexactproteindeterminationwithalbuminasastandard,sincethisproteincontainsmorebasicaminoacidsthanmanyotherproteins.TheredoexistmanyprotocolsforsensitivestainingprocedureswithCoomassie®(e.g.ref.3,4).Thesensitivityreachesalimitat25ngprotein.Werecommendthefollowingprotocol:I. Staining solution: 0.1 % Coomassie® Brilliant Blue R-250 (Prod.-No. A1092), 20 % methanol (or ethanol), 10 % acetic acid.TheSDSgel(without'stackinggel')isstainedfor1hourat60°Corfor2hoursat50°CorovernightatRT.
II. Destainingsolution:20%methanol(orethanol)and10%aceticacid.Destainthegel for3-4hoursat50-60°C.Addsomesponges.Subsequentlywashthegelfor15minutsinwateranddryundervacuumat60°Cfor2-3hours.
4.1
4.2
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els
4.2
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els
*Allen,R.E.et al.(1980)Anal. Biochem. 104,494-498.StainingProteinsinIsolelectricFocusingGelswithFastGreen.**stainse.g.sialoglycoproteinsandphosphoproteinsblue,almostallotherproteinsred.
©2010AppliChem•SizeMarker 25
Prod.-No. Description Comment Absorption maxima Sensitivity Recommended Concentration Stock solution
A2176 BismarckbrownR increasessensitivityofCoomassie® BrilliantBlueR-250staining
lmax.468nm 25ng& CoomassieBrilliantblueR-250(0.2%w/v)BismarckbrownR(0.05%w/v)
SolventMeOH:Aceticacid:Water(40:7:53); dissolvedyesseparately,thenmix1:0.75(v/v)
A3480 Coomassie®BrilliantBlueG-250 stainsampholytesinIEFgelstoo!* <0.5ng Mix80mlofthestocksolution with20mlofMeOHjustbeforeuse.
0.1%w/vCBBin2%w/vPhosphoricaicd, 10%w/vAmmoniumsulfate(Donotfilter!)
A1092 Coomassie®BrilliantBlueR-250 stainsampholytesinIEFgelstoo!* lmax.withprotein549nm, w/oprotein555nm
10ng 0.1%in20%MeOH,10%Aceticacid
A0822 EosinY reversiblestaining similartoCBBR-250 0.25%(w/v)in0.1NNaOH preparestainingsolutionfreshdaily; maybeusedforseveralgels;
A1346 EriochromeblackT 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid; incombinationwithRhodamineB
A1401 FastGreenFCF dyecomplexinacidicpHrange lmax.(pH8.3)615;(MeOH)620nmlmax.(withprotein)635nm
400ng 0.25%(w/v)in10%Aceticacidupto1%(w/v)in7%Aceticacid
A2385 Kongored stainingintheacidicpHrange lesssensitivethanCBB 0.1%(w/v)in0.2MAcetatebuffer(pH3.5) 1%(w/v)indistilledwaterA2388 Malachitegreenoxalate
Phosphatasestainingingels
Stainingsolution= 3Vol.Solution1+1Vol.Solution2 (stablefor3weeks;filterpriortoeachuse)
Solution1:0.15gMalachitegreenoxalatein300mlWater;Solution2:4.2gAmmoniummolybdatetetrahydratein100ml5NHCl;
Nilered 5ngA1405 PonceauS acidicdiazodye lmax.(H2O)517-823nm 500ng 0.1-0.5%in3%TCAor1mlAceticacid
glacialad100mlWaterpreparefreshpriortouse
A7808 Proteo-DyeRuBPS lmax.617nm 2-5ng 1µM 1mMA3930 RhodamineB$ 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid;incombinationwith
EriochromeblackTA3972 Silvernitrate brown 2-5ngA1400 Stainsall** stainsdifferenttypesofproteins
indifferentcolorslmax.forBSA515nm likeCBB 0.005% 0.1%inFormamideor5.6mg/10mlin50%Dioxane;
preparefresh
Zinc-Imidazole reversiblestaining 10-20ngSDS-PAGE 40-80ngnativePAGE
Coomassie® Brilliant blue G-250 (C.I. 42655) A3480Synonym BrilliantblueG
Order No. Quantity Solubility (20°C) Formula M CAS-No.
A3480,0010 10g ~10g/L(H2O) C47H48N3NaO7S2 854.04g/mol 6104-58-1
A3480,0025 25g
A3480,0100 100g
®registeredtrademarkofImperialIndustriesPLC
TheassaysofNeuhoffetal.(Neuhoff,V.etal.(1988)Electrophoresis9,255-262)haveshownthatstainngwithCoomassie®G-250ismoresensitivethanwithR-250.FormoreinformationsseeA1092.
26 SizeMarker•©2010AppliChem
dyes
Protocol for staining of gels with Proteo-Dye Blue-Vis: SDS-PAGEminigels (1mm thickness, 10% acrylamide, Tris-glycine buffer system) aretreatedfor1hourwith30%v/vmethanolafterelectrophoresis toremovetheexcessofSDSandtofixtheproteinsinthegelmatrix.Thegelsarestainedfor2hoursinavolumeof100mlProteo-DyeBlue-Vis.Afterthis,thegelsarewashedwithacetatebuffer(0.2M;pH4.5),containing20%v/vmethanolfor90-120minutesuntilthedarkredbackgroundisreducedtoaweakpinkbackgroundincontrasttotheblueproteinbands.Thegelsaredocumentedwiththehelpofavideosystem(transilluminator312nm,whitelightplate)oronascannerinthetransmissionmode.
Proteo-Dye Blue-Vis A6810
Synomym Proteindyeinthevisiblerange
Storage 2-8°Cprotectedfromlight
HS-No. 38220000
Package size A6810,1000 1L
1Lissufficientforapprox.30minigels,since3xreusable detectionlimit3-5ng/mm² reversiblestainingofproteins
Protocol for staining of gels with Proteo-Dye Red-Fluo: SDS-PAGEminigels(5x8cm,1mmthickness,10%acrylamide,Tris-glycinebuffersystem)arefixedfor30minutesinasolutionof7.5%v/vaceticacid,20%v/vethanol.Afterthis,thegelsarestainedwithProteo-DyeRed-Fluo(100ml)for2-3hours.Thebackgroundfluorescence canbe removedby repeatedwashingwith the fixation solution (7.5% v/vaceticacid,20%v/vethanol;3-4washingsteps,30minuteseach).Thedocumentationof the gels is carried out with a video system (transilluminator 312 nm, adapting filter590nm).
Proteo-Dye Red-Fluo A6803
Synomym Proteindyewithredfluorescence
Storage 2-8°Cprotectedfromlight
HS-No. 38220000
Specification Emissionmaximum:630nm
Exicitationwavelenght:312nm
Package size A6803,1000 1L
1Lissufficientforapprox.30minigels,since3xreusable detectionlimit1-3ng/mm² reversiblestainingofproteins moresensitivethanmostotherproductsavailableinthemarket
4.3
©2010AppliChem•SizeMarker 27
for protein gels
Proteo-DyeGreen-Fluoisafluorescenceproteindye,basedonametal-chelatecomplexinconjunctionwithadetergentinsubmicellarconcentrationinanaqueoussolution.Thisdyesolutionenablestodetectverysmallamountsofproteinsandthestainingisfullyreversible.Usethedyesolutionuptothreetimes!
Protocol for staining of gels with Proteo-Dye Green-Fluo: SDS-PAGEminigels(5x8cm;1mmthickness,10%acrylamide,Tris-glycinebuffersystem)arepostelectrophoretically treated for30minuteswithamethanol solution(30%, v/v)whilegentlyshaking(100rpm).Afterthis,thegelsarestainedfor2hoursinavolumeof100mlProteo-DyeGreen-Fluo.Reductionofthebackgroundfluorescenceisachievedbyrepeatedwashingsteps(3-4 times,30min.each)with30%v/vmethanol.Thegelsaredocumentedwithavideosystem(transilluminator365nm,adaptingfilter520nm).
Protocol for staining of blots with Proteo-Dye Green-Fluo: BystainingwithCoomassieorsilvertheproteinsinthegelbecomeirreversiblydenaturatedand are difficult to transfer to blottingmembranes.Metal chelate stainingmethods arereversible, resulting in good preconditions for the following blot transfer. Blots wereperformedbyatypicalsemi-drymethod.Aftertheblottingprocess,theblotswerewashedindistilledwaterfor1houranddriedatroomtemperature.BlotswerethenstainedwithProteo-DyeGreen-Fluo for2hours,brieflywashed in30%v/vethanolanddriedagain.Only in thedried state, intenselygreen fluorescentproteinbandsbecomevisible inUVtoplight(365nmUVlight)andaredocumentedwithavideosystem(adaptingfilter420nm).Thestainmaybeeasilyandfastlyremovedjustbyrinsingin30%v/vethanol.
Proteo-Dye Green-Fluo A6794
Synomym Proteindyewithgreenfluorescence
Storage 2-8°Cprotectedfromlight
HS-No. 38220000
Specification Emissionmaximum:520nm
Excitationwavelenght:365nm
Package size A6794,1000 1L
1Lissufficientforapprox.30minigels,since3xreusable detectionlimit3-5ng/mm² reversiblestainingofproteins suitableforgelsandforblots
28 SizeMarker•©2010AppliChem
CommentRuBPSisafluorescencedyeforproteindetectionine.g.SDS-and2-D-gels.Itprovidesthehighsensitivity of silver staining without its drawbacks. The excellent contrast, good linearity andhomogeneitymadethisdyethestainingreagentofchoiceinproteomics.AminimalinterferencewithMALDI-TOFanalysisisobservedandcompatibilitywithMS/MSanalysisisgiven.Thephoto-chemicallystabledyeisexcitedwithUV-lightofthewavelength473or488nmbluelaser.A532nmlasermaybeusedaswell,albeitwithreducedefficiency.ItisofadvantagethattheexcitationwavelengthofRuBPSislongerthantoseofthearomaticaminoacids.The information given in terms of formula, molecular weight and CAS number refer to theraw material! The concentrate supplied is diluted 1 : 1000 (1 ml ad 1 L) resulting in an 1µMworkingsolution.
Special features of Proteo-Dye RuBPS• Providesthesensitivityofsilver-stainingwithoutitsdrawbacks (limiteddynamicrange,inhomogeneityandinterferencewith MSanalysis)• Carefullypurifiedproductoffersmuchimprovedsignal-to- backgroundratio• Excellentcontrastingelimagesenablesproteinspotstobe detectedmoreeasilybyimageprocessingsoftware• Highsensitivity• Goodlinearityandhomogeneity• MinimalinterferencewithMALDI-TOFanalysis• CompatiblewithMS/MSanalysis,providedthatcleaningofthe samplewithreversephaseadsorptioniscarriedout
Protocol for gel staining / destaining(accordingtoRef.2)1. Prepare a 1 µM Proteo-Dye RuBPS solution by diluting the concentrate 1000-fold (e.g.,1mlto1L)
2. Fixthegelin30%ethanol,10%v/vaceticacidovernight.3. Rinsethegelin20%ethanolv/vfor30minandrepeat3times.4. Incubatethegelin1µMProteo-DyeRuBPSsolutionfor6h.5. Equilibrate the gel in water for 10 min and repeat once (a first scan is possible at thisstage).
6. Destainthegelwith40%ethanol/10%aceticacidv/vfor15h.N.B. for low protein concentration the destaining time might be too long. In such cases, shor-ter destaining times may optimize the procedure resulting in greater sensitivity.7. Equilibratethegelinwaterfor10min,repeatonceandscan.
Proteo-Dye RuBPS A7808
Synomym Ruthenium(II)tris(bathophenanthrolinedisulfonate)tetrasodiumsalt
Formula C72H42N6Na4O18RuS6
Molecularweight 1664.58g/mol
CAS-No. [301206-84-8]
Concentration(inH2O) 1mM
lmax.Emission(inH2O) 617nm
lmax.Exitation 277,437,460nm
Storage 2-8°C,protectedfromlight
recommendedworkingsolution 1:1000dilution(1µMfinalconcenrtation)
Stability 2years
Packagesize A7808,0001 1ml
2-D gels of E. coli proteins stained with (1) SYPRO Ruby© and (2) with RuBPS. For details see Reference 2.
Literature
[1] AcomparisonbetweenSyproRuby©and
ruthenium(II)tris(bathophenanthrolinedisulfonate)
asfluorescentstainsforproteindetectioningels.
(Rabilloud,T.etal.(2001)Proteomics1,699-704)
[2] Improvedruthenium(II)
tris(bathophenanthrolinedisulfonate)staining
anddestainingprotocolforabettersignal-to-
backgroundratioandimprovedresolution.
(Lamanda,A.etal.(2004)Proteomics4,599-608)
©2010AppliChem•SizeMarker
Electrophoresis Reagents Prod. No.AcrylamideMolecularbiologygrade A3812
BisacrylamideMolecularbiologygrade A3636
AgaroseBasic A8963
AgaroselowEEO(AgaroseStandard) A2114
AgaroseMP A1091
LoadingbufferDNAI A3144
LoadingbufferDNAII A2571
LoadingbufferDNAIV(forAgarosegels) A3481
TAEbuffer(50X) A1691
TAEbuffer(10X) A1416
TBEbuffer(10X) A0972
Transfer membranes Prod. No.ReprobeNitrocellulosesupported0.22µmTransfermembrane A5237
ReprobeNitrocellulosesupported0.45µmTransfermembrane A5242
PureNitrocelluloseunsupported0.22µmTransfermembrane A5250
PureNitrocelluloseunsupported0.45µmTransfermembrane A5239
PureNylonNeutralTransfermembrane0.22µm(30cmx3m) A4399
PureNylonNeutralTransfermembrane0.45µm A5248
ReprobeNylonPositivelychargedTransfermembrane0.45µm(30cmx3m) A5255
PVDF-StarTransfermembrane0.45µm A5243
Other Related Products Prod. No.ChemiluminescenceDetectionKitsforHorseradishPeroxidase
Cheluminate-HRPPicoDetect A3417
Cheluminate-HRPFemtoDetect A7807
Cheluminate-HRPFemtoDetectPlus A7879
BoricacidMolecularbiologygrade A2940
EDTAdisodiumsaltdihydrateMolecularbiologygrade A2937
Trisultrapure A1086 rela
ted
prod
ucts
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