In-Gel Digestion Why In-Gel Digest? Difficult / impossible to extract intact proteins from the gel
Gel electrophorosis final
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Transcript of Gel electrophorosis final
Introduction Introduction Gel electrophoresis is a method used
in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix.
Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field.
The gel acts as a sieve to retard the passage of molecules according to their size and shape.
Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving
Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
Agarose gels are easily cast and handled
compared to other matrices, because the gel setting is a physical rather than chemical change. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.
advantages: it is used for the separation of DNA fragments ranging from 50 base pair to several millions of bases using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel
The disadvantage of higher concentrations is the long run times (sometimes days).
Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly
Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger
PolyacrylamidePolyacrylamidePolyacrylamide gel
electrophoresis (PAGE) is used for separating proteins
Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.
StarchStarchPartially hydrolyzed potato starch
makes for another non-toxic medium for protein electrophoresis.
The gels are slightly more opaque than acrylamide or agarose.
Non-denatured proteins can be separated according to charge and size.
They are visualized using Napthal Black or Amido Black staining.
How does it work?How does it work?
• DNA is cut into smaller fragments.
• Loading dye is used to indicate the fragments of DNA are behind the dye
• The negative DNA molecule is attracted to the positive electrode.
• The smallest fragments move the greatest distance.
Gel electrophoresisGel electrophoresisA method of separating
DNA in a gelatin-like material using an electrical field◦DNA is negatively charged◦when it’s in an electrical
field it moves toward the positive side
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DNA
“swimming through Jello”[email protected]
Gel electrophoresisGel electrophoresisDNA moves in an electrical field…
◦so how does that help you compare DNA fragments? size of DNA fragment affects how far it
travels small pieces travel farther large pieces travel slower & lag behind
+–
DNA
“swimming through Jello”
Gel ElectrophoresisGel Electrophoresis
longer fragments
shorter fragments
powersource
completed gel
gel
DNA &restriction enzyme
wells
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Running a gelRunning a gel
Stain DNA◦ ethidium bromide
binds to DNA◦ fluoresces under
UV light
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cut DNA with restriction enzymes
fragments of DNAseparate out based on size
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ProcedureProcedureRemove comb and observe wells.Place carbon paper in each end of the
tray.Cover with buffer, making sure the allow
buffer to overflow into each end of the tray.
Load gels.Connect the electrodes.Turn on power supply.Allow gels to run – make sure you see
bubbles coming from the [email protected]
PROCEDURE (CONTINUED)PROCEDURE (CONTINUED)It will take about 30 minutes for
the gel to run.Turn off power supply and
remove electrodes.Pour off buffer into the
designated container.Carefully remove gel from gel
box and place in glad container and cover with stain.
Store in appropriate location.
ApplicationsApplicationsEstimation of the size of DNA
molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Separation of restricted genomic DNA Gel electrophoresis is used
in forensics, molecular biology, genetics, microbiology and biochemistry.
Proteins can also be run on gels. Most commonly proteins are run on gels made of polyacrylamide in the presence of SDS
Scientific dyes can also be separated by gel electrophoresis.
