Estimation of total free amino acids

The amino acids are colorless ionic compounds that form the basic building blocks of protein. Apart from being bound as proteins, amino acids also exist in the free form in many tissues and are known as free amino acids. They are mostly water soluble in nature. Very often in plants during disease conditions, the free amino acid composition exhibits a change hence, the measurement of the total free amino acids gives the physiological and health status of the plants. PrincipleNinhydrin, a powerful oxidizing agent, decarboxylates the alpha-amino acids and yields an intensely colored bluish purple product which is colorimetrically measured in 570nm. Ninhydrin + alpha-amino acid

Hydrindantin + Decarboxylated Amino acid + Carbon dioxide + Ammonia

Hydrindantin + Ninhydrin + Ammonia

Purple colored product + Water

  Materials Ninhydrin: dissolve 0.8g stannous chloride (SnCl2.2H2O) in 500mL of 0.2M citrate buffer (pH 5.0). add this solution to 20g of ninhydrin in 500mL of methyl cellosolve (2 methoxyethanol) 0.2M Citrate Buffer pH 5.0 Diluent Solvent: Mix equal volumes of water and n-propanol, and use.

ProcedureExtraction of Amino AcidsWeigh 500mg of plant sample and grind it in a pestle and mortar with a small quantity of acid-washed sand. To this homogenate, add 5to 10mL of 80% ethanol. Filter or centrifuge. Save the filtrate or supernatant. Repeat the extraction twice with the residue and pool all the supernatants. Reduce the volume if needed by evaporation and use the extract for the quantitative estimation of total free amino acids. If the tissue is tough, use boiling 80% ethanol for extraction.

Estimation1.

To 0.1mL of extract, add 1mL of ninhydrin solution.

2.

Make up the volume to 2mL with distilled water.

3.

Heat the tube in boiling water bath for 20min.

4.

Add 5mL of the diluents and mix the contents.

5.

After 15min read the intensity of the purple color against a reagent blank in a colorimeter at 570nm. The color is stable for 1h.

6.

Prepare the reagent blank as above by taking 0.1mL of 780% ethanol instead of the extract.

 

StandardDissolve 50mg leucine in 50mL of distilled water in a volumetric flask. Take 10mL of this stock standard and dilute to 100mL in another flask for working standard solution. A series of volume from 0.1 to 1mL of this standard solution gives a concentration range 10g to 100g. proceed as that of the sample and read the color. 

ResultDraw a standard curve using absorbance versus concentration. Find out the concentration of the total free amino acids in the sample and express as percentage equivalent of leucine. 

Notes1. Ninhydrin is carcinogenic. Wear gloves while handling it.2. Since this estimation includes only alpha-amino acids, non protein amino acids are not accounted. 

References 1. Moore, S and Stein, W H (1984) In: Methods in Enzymol (Eds. Colowick, S P and Kaplan, N D) Academic Press New York 3 468.2. Misra, P S, Mertz E T and Glower, D V (1975) Cereal Chem 52 844.3. Theymoli Balasubramanian and Sadasivam, S (1987) Plant Food Hum Nutr  37 41.

IDENTIFICATION OF PROTEINS:

Identification of proteins Proteins are present in the living world, irrespective of the size of the organism, since they form the structural and functional basis of cell. Under certain circumstances, it may become necessary to identify the presence of protein. Isolation of any new compound (e.g., antiviral principle) needs to be attacked primarily through identification reactions. Some of the color reactions of proteins which could be used as identification tests are given below: 

Test Observation Remarks1. BIURET REACTION

To 2mL of the test solution add 2mL of 10% NaOH. Mix. Add two drops of 0.1% CuSO4 solution

Violet or pink color Compounds with two or more peptide bonds give a violet color with alkaline copper sulphate solution

2. NINHYDRIN TESTTo 4mL of the solution which should be at neutral pH add 1mL of 0.1% freshly prepared ninhydrin solution. Mix the contents and boil for a couple of minutes. Allow to cool

Violet or purple color The ninhydrin test is answered by amino acids and proteins. The formation of a complex called Rheumann’s purple due to condensation of two molecules of nin-hydrin with one molecule of ammonia from amino acid is responsible for the violet color. The -amino group is the reactive group

3. XANTHOPROTEIC REACTIONTo 5mL of the solution add 1mL of conc. HNO3. Boil the contents. After cooling ad excess 40% NaOH

On adding acid, yellow color will be noticed. When NaOH is added deep orange color will develop

The yellow color is due to the nitro derivatives of the aromatic amino acids present in the protein. The sodium salts of nitro derivatives are orange in color.

4. GLYOXYLIC REACTION FOR TRYPTOPHAN(Hopkins-Cole test)Add 2mL of glacial acetic acid to 2mL of the test solution. Then about 2mL of conc. H2SO4 carefully down the sides of the test tube. Observe the color change at the junction of the two liquids.

Violet ring is formed at the junction

The indole group of tryptophan reacts with the glyoxylic acid released by the action of conc. H2SO4

on acetic acid to give a purple color.

5. SAKAGUCHI REACTIONTo 5mL of the solution cooled on ice add 1mL of 10% NaOH solution and 1mL of 0.02% -Naphthol solution. After few minutes add 10 drops of alkaline hypobromide solution

Intense red color The guanidine group or arginine reacts with -Naphthol to form a bright red colored complex.

6. SULPHUR TESTTo 2mL of solution add 2mL of 40% NaOH and 10 drops of 2% lead acetate solution. Boil for a minute and cool.

Black precipitate The sulphur in sulphur containing amino acids of the proteins in presence of NaOH, is changed into Na2S which forms black lead sulphide when reacted with lead acetate.

7. MODIFIED MILLION’S TESTa) To 1mL of solution add 1mL of

Yellow precipitate The yellow precipitate is due to

10%mercuric sulphate in 10% sulphuric acid. Boil gently for half a minute.

the precipitation of protein. Mercury combines with tyrosine of the protein.

b) Cool under a tap water and add a drop of 1% NaNO2 solution and warm gently

A red color develops The red color is due to reaction of the precipitate with the nitrous acid.

Polyacrylamide-sodium dodecyl sulphate slab gel electrophoresis (SDS-PAGE) of proteins Electrophoresis is widely used to separate and characterize proteins by applying electric current. Electrophoretic procedures are rapid and relatively sensitive requiring only micro-weights of proteins. Electrophoresis in the polyacrylamide gel is more convenient than in any other medium such as paper and starch gel. Electrophoresis of proteins in polyacrylamide gels is carried out in buffer gels (non-denaturing) as well as in sodium dodecyl sulphate (SDS) containing (denaturing) gels. Separation in buffer gels relies on both the charge and size of the protein whereas it depends only upon the size in the SDS-gels. Analysis and comparison of proteins in a large number of samples is easily made on polyacrylamide gel slabs.

Polyacrylamide gels are formed by polymerizing acrylamide with a cross-linking agent (bisacrylamide) in presence of a catalyst (persulphate ion) and chain indicator (TEMED; N,N,N’,N’ – tetramethylethylene diamine). Solution are normally degassed by evacuation prior to polymerization since oxygen inhibits polymerization. The porosity of the gel is determinedly the relative proportion of acrylamide monomer to bisacrylamide.Gels are usually referred to in terms of the total percentage of acrylamide and bis present, and most protein separations are performed using gels in the range 7-15%. A low percentage gel (with large pore size) is used to separate high molecular weight proteins and vice-versa. At high concentrations of persulphate and TEMED the rate of polymerization is also high. Among a number of methods commonly used, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in slabs (facilitating characterization of polypeptides and determination of their molecular weight by co-electrophoresis) is described below.  PrincipleSDS is an anionic detergent which binds strongly to, and denatures, proteins. The number of SDS molecules bound to a polypeptide chain is approximately half the number of amino acid residues in that chain. The protein-SDS complex carries net negative charges, hence move towards the anode and the separation is based on the size of the protein.  Materials Stock Acrylamide SolutionAcrylamide 30%                                                         30gBisacrylamide 0.8%                                                    0.8g

Water to                                                                     100mL  Separation Gel Buffer 1.875M Tris-HCl                                                          22.7g   pH 8.8Water to                                                                    100mL  Stacking Gel Buffer0.6M Tris-HCl7.                                                            26g   pH 6.8Water to                                                                     100mL  Polymerizing Agentsa) Ammonium persulphate 5%                                0.5g/10mL, prepare freshly before useb) TEMED                                                                 fresh from the refrigerator  Electrode Buffer0.05M Tris                                 12g0.192M Glycine                          28.8g   pH 8.2-8.40.1% SDS                                  2g No adjustment requiredWater to                                    2mLmay be used 2-3 times  Sample Buffer (5X concentration)Tris-HCl buffer pH 6.8                 5mLSDS0.                                         5gSucrose                                      5gMercaptoethanol0.                     25mLBromophenol Blue                      1mL(0.5% W/V solution in water)Water to                                    10mLStore frozen in small aliquots. Dilute to 1x concentration and use.  Sodium dodecyl sulphate 10% solution – store at room temperature.  Standard Marker ProteinsProtein                                                                       MW Daltonsa-Lactalbumin                                                           14,200Trypsin inhibitor soyabean                                        20,100Trypsinogen                                                              24,000Carbonic anhydrase                                                  29,000Glyceraldehyde-3-phosphate dehydrogenase, rabbit                                                                        36,000Albumin, egg                                                             45,000Albumin bovine                                                         66,000Dissolve the above proteins in single strength sample buffer at a concentration each of 1mg per mL. Load the well with 25-50L.   Protein Stain SolutionCoomassie brilliant blue R 250                                0.1gMethanol                                                                       40mLAcetic acid                                                                    10mLWater                                                                             50mL

First, dissolve the dye in methanol and proceed. Use fresh preparation every time.  DestainerAs above without the dye.  Procedure1. Thoroughly clean and dry the glass plates and spacers, then assemble them property. Hold the

assembly together with bulldog chips. Clamp in an upright position. White petroleum jelly or 2% agar (melted in a boiling water bath) is then applied around the edges of the spacers to hold them in place and seal the chamber between the glass plates.

2. Prepare a sufficient volume of separating gel mixture (30mL for a chamber of about (18 x 9 x 0.1cm) by mixing the following.

for 15% gel for 10% gelStock acrylamide solution 20mL 13.3mLTris-HCl (pH 8.8) 8mL 8mLWater 11.4mL 18.1mLDegas on a water pump for 3 – 5min and then add:Ammonium persulphate solution

0.2mL 0.2mL

10% SDS 0.4mL 0.4mLTEMED 20L 20L

3. Mix gently and carefully, pour the gel solution in the chamber between the glass plates. Layer distilled water on top of the gel and leave to set for 30-60min.

4. Prepare stacking gel (4%) by mixing the following solutions (total volume 10mL)Stock acrylamide solution = 1.35mLTris-HCl (pH 6.8) = 1mLWater = 7.5mLDegas as above, and then add:Ammonium persulphate solution (5%) = 50L10% SDS = 0.1mLTEMED = 10LRemove the water from the top of the gel and wash with a little stacking gel solution. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set (30-60min).

5. After the stacking gel has polymerized, remove the comb without distorting the shapes well. Carefully install the gel after removing the clips, agar etc. in the electrophoresis apparatus. Fill it with electrode buffer and remove any trapped air bubbles at the bottom of the gel. Connect the cathode at the top and turn on the DC-power briefly to check the electrical circuit. The electrode buffer and the plates can be kept cooled using a suitable facility so that heat generated during the run is dissipated and does not affect the gel and resolution.

6. Prepare samples for electrophoresis, following suitable extraction procedure. Adjust the protein concentration in each sample using the 5-strenght sample buffer and water in such a way that the same amount of protein is present per unit volume. Again the concentration should be such as to given a sufficient amount of protein (50-200g) in a volume (25-50L) not greater than the size of the sample well. As general practice, heat sample solution in boiling water for 2-3min to ensure complete interaction between protein and SDS.

7. Cool the sample solution and take up the required volume in a microsyringe and carefully inject it into a sample well through the electrode buffer. Making the position of well on the glass plate with a marker pen and the presence of bromophenol blue in the sample buffer facilitate easy loading of the

samples. Similarly, load a few well with standard marker protein in the sample buffer.8. Turn on the current to 10-15mA for initial 10-15min until the samples travel through the stacking gel.

The stacking gel helps concentration of the samples. Then continue the run at 30mA until the bromophenol blue reaches the bottom of the gel (about 3h). however, the gel may be run at a high current 960-70mA) for short period (1h) with proper cooling.

9. After the run is complete, carefully remove the gel from between the plates and immerse in staining solution for at least 3h or overnight with uniform shaking. The proteins absorb the coomassie brilliant blue.

10.

Transfer the gel to a suitable container with at least 200-300mL destaining solution and shake gently continuously. Dye that is not bound to proteins is thus removed. Change the destainer frequently, particularly during initial periods, until the background of the gel is colorless. The proteins fractionated into band are seen colored blue. As the proteins of minute qualities are stained faintly, destaining process should be stopped at appropriate stage to visualize as many bands as possible. The gel can now be photographed or stored in polythene bags or dried in vacuo for a permanent record (for details see ‘Flurography of polyacrylamide gels’).

   Notes1. All chemicals and distilled water should be of high quality. The solutions prepared should be filtered

before use. The solutions can be stored refrigerated for 1-2 weeks; aged solution result in poor resolution of proteins.

2. Acrylamide as a monomer is highly neurotoxic; handle with extreme care.3. Prefer to use the gel immediately following polymerization although the separation gel after setting

can be stored overnight by wetting with four-fold diluted separation gel buffer or with stacking gel and comb placed over it to avoid drying.

4. Degassing of gel mix should be adequate for easy polymerization.5. During polymerization of the gel, heat is evolved.6. The water layered over the separation gel should be completely removed for quick polymerization of

the stacking gel.7. Some troubles and remedies are as follows:

Trouble Cause Remedya. Failure or slow

polymerization of the gel

Presence of oxygen

Absence of catalysts

Stock aged Glass plates

Degas the solution sufficiently

Check if all solution mixed

Use fresh solutions Degrease the plates

with ethanol

b.

Poor sample wells Stacking gel and comb

Fit and/or remove the comb carefully.

c. Long duration of the run

Air-bubbles interference

Flush air-bubbles

d.

Staining is poor The dye absorption is not efficient

The dye may be old, hence use a strong solution of the dye or change to a more sensitive stain

The staining is patchy Solid dye Dissolve the dye completely or filter

The stained bands are decolorized

The dye is removed excessively

Restain the gel and stop destaining appropriately

e. Protein bands are inadequately resolved

Insufficient electrophoresis

Run for longer time

Separation gel Change the percentage of gelf. Protein bands are

wavyExcess persulphate Use optimum concentration

of presulphateBands have become streaked

Proteins remain aggregated, denatured or insoluble

Use fresh sample buffer or extra SDS or centrifuge the sample extract sufficiently

g.

Protein dye migration is not even

Gel is partly insulated by air bubbles.

Remove air bubbles before electrophoresis

Insufficient cooling Improve the cooling or run at a low current

h.

The protein band-lane broadens at the bottom of the separation gel

Sample density Load equal volume of samples in each well, equal strength sample buffer, leave no empty wells in the middle

Sample diffuses while loading the wells

Low density of sample

Increase the concentration of sucrose or glycerol in the sample buffer

8. Handle the polyacrylamide gel carefully to avoid any breakage.9. The slab gel along the glass plates is placed vertically in the electrophoresis tank and run. It is

therefore called ‘vertical slab gel electrophoresis’.10.

In 10% polyacrylamide gels, the low molecular weight (~10,000 daltons) polypeptides will migrate diffused; for fine resolution of these polypeptides use gels of higher (15%) acrylamide concentration.

11.

Any band of 0.1g protein is visualized by coomassie brilliant blue staining in SDS-PAGE; for visualizing proteins of lower concentration below 0.1g high sensitive (silver staining) method is prepared.

  

References1. Laemmli, U K (1970) Nature 227, 680.2. Manual on Techniques in Molecular Biology (Proteins) (1986). Workshop held at the Department of Biochemistry Tamil Nadu Agri Univ Coimbatore p 16.

Protein Estimation – Bradford Method The protein in solution can be measured quantitatively by different methods. The method described by Bardford uses a different concept – the protein’s capacity to bind a dye, quantitatively. The method is simple, rapid and inexpensive.

 

PrincipleThe assay is based on the ability of proteins to bind coomassie brilliant blue G 250 and form a complex whose extinction coefficient in much greater than that of the free dye.  Materials Dye concentrateDissolve 100mg of coomassie brilliant blue G 250 in 50mL of 95% ethanol. Add 100mL of conc. (ortho) phosphoric acid. Add distilled water to a final volume of 200mL. store refrigerated in amber bottles; the solution is stable at least 6 months. Mix 1 volume of concentrated dye solution with 4 volumes of distilled water for use. Filter with Whatman No. 2 if any precipitate occurs. Phosphate-buffered saline (PBS).  Procedure1. Prepare a series of protein samples in test tubes in the concentration. This is preferably prepared in

PBS.2. Prepare the experimental samples (a few dilutions) in 100L of PBS.3. Add 5mL of dilute dye binding solution to each tube.4. Mix well and allow the color to develop for at least 5min but no longer than 30min. the red dye turns

blue when it binds protein.5. Read the absorbance at 595nm.6. Plot a standard curve using the standard protein absorbance Vs concentration. Calculate the protein in

the experimental sample using the standard curve.  Notes1.

It is important to use a protein as similar in its properties to your sample as possible! If your sample is unknown, use antibody protein as reference. BSA usually gives a 2-fold higher value in this assay and therefore cannot be used as a general standard.

2.

As in the case of Lowry’s protein assay procedure detergents such as SDS, Nonidet P40, Triton x 10 etc. interfere with this protocol, too.

3.

Serva blue G dye is another dye used in place of coomassie blue G 250 with similar properties.

4.

The dyes exist as two forms (blue and orange) in acid solution. The proteins bind the blue form preferentially.

5.

Check the absorption of working dye solution at 550nm is 1.18, if necessary, adjust either with the powder or water as required.

  References Bradford, M M (1976) Anal Biochem 72.

Isoelectric focusing Isoelectric focusing (IEF) is an electrophoretic method for the separation of proteins according to their isoelectric points (pI). It is reproducible, sensitive and highly useful to resolve closely related proteins which may not be so well separated by other techniques.

  PrincipleAnalytical IEF is carried out in a thin layer of polyacrylamide gel containing a large series of carrier ampholytes. When a potential difference is applied across the gel, the carrier ampholytes align themselves in order of increasing pI from the anode to the cathode, thus producing a uniform pH gradient across the gel. Under the influence of the electric field each protein migrates to the region in the gradient where the pH corresponds to its pI. The protein is electrically neutral at its pI and where it gets focused in the gel. After focusing, the separated components are detected by appropriate staining.  Materials Acrylamide, bisacrylamide and sucrose, all analytical grade. Riboflavin solution, 1mg/10mL. store refrigerated in brown bottle. Carrier ampholytes of the suitable pH range (pH 3-10, pH5-7 or pH 4-8) store at 4°C. Glass plates 20 x 15 x 0.4cm dimension. Fixing Solution: 5g sulphosalicylic acid and 10g trichloroacetic acid (TCA) in 90mL distilled water. Destaining Solution: Methanol: acetic acid: water in the ratio 3:1:6 (v/v) Staining Solution: 0.2% coomassie brilliant blue R250 in the destaining solution. Filter before use. Wick Solutions: 1M phosphoric acid for anode and 1M NaOH for cathode. Electrofocusing system.  Procedure1. Stick strips of insulating tape 1cm wide and approximately 0.20mm thick around the edge of a clean

glass plate. This shall give a very shallow tray of 18 x 13 x 0.015cm in which a thin polyacrylamide gel is cast.

2. Dissolve the following components completelyAcrylamide                           1.94gBisacrylamide                      0.06gSucrose                                 5.0gRiboflavin solution              0.25mLWater                                     40mL

3. Add 2mL of carrier ampholyte solution of the appropriate pH range to the above mix. Ensure that all the components are uniformly mixed by gently swirling the flask until poured. The entire solution will be sufficient for six plates.

4. Place a glass mold in large tray with the taped surface uppermost. Wipe clean the surface with an alcohol moistened tissue or remove any traces of grease.

5. Transfer approximately 7mL of the above solution to one end of the glass mold.6. Place a clean plain glass plate (20 x 15cm) one of the short edges along the taped edge of the mold

adjacent to the acryamide-ampholyte solution. Gradually lower the top plate and allow the solution to spread over the mold without entrapping any air bubbles. Press the top plate into firm contact with the taped edge of the bottom plate.

7. Lift the complete mold and top plate out of the large tray. Remove any gel material at the edges and bottom of the mold plate.

8. Photopolymerize the gel for at least 3h under white light or bright direct sunlight to provide sufficient UV light.

9. After polymerization wipe the outside surfaces with a wet tissue to remove any solid material, otherwise it may affect cooling during the run.

10.

The plates may be stored for a month in the dark at 4°C. the removal of the top plate is easier when cooled at 4°C for at least overnight.

11.

Prior to sample application, remove the top plate carefully, inserting a scalpel blade between the two glass plates at the corner. The whole gel should stick either at the bottom or top plate for use. Occasionally, part of the gel will stick to the top plate and part to the bottom plate. In such cases, the gel has to be discarded.

12.

Absorb sample solutions (5-8L) on 5 x 5mm pieces of Whatman No. 1 filter paper and lay these on the gel surface along the length of the plate at about 5cm from the cathode edge. The protein concentration between 0.05 and 0.15mg is generally sufficient.

13.

Place the gel plate on the cooling plate of the elctrofocusing apparatus through which water at 4-8°C is circulated.

14.

Apply electrode wicks (strips of Whatman 17 filter paper) to the anode and cathode edges of the gel. The anode and cathode wicks are uniformly soaked in 1M phosphoric acid and 1M NaOH respectively before being placed. (instead, 1% acetic acid and 1% ethanolamine may also be used as a wick solutions).

15.

Apply a potential difference of between 130 and 160 V/cm across the plate. Focusing takes 2-4h. focusing is complete when the same components of the same sample is placed on the cathode and anode sides in parallel lanes reached similar zones.

16.

When focusing is complete, disconnect the power supply, remove the electrode and wicks and lift the gel plate from the tank carefully.

17.

Place the gel plate in the fixing solution for 15-20min. transfer the plate to the destaining solution for 15min. then transfer the gel to staining solution for 30min; and finally to destaining solution for about 20min until the protein bands are clearly visible. The entire process is done at room temperature.

18.

To preserve the gel, first immerse the destained gel in destaining solution containing 10% glycerol for 30-60min. soak a cellophane sheet in the same solution for a few minutes and wrap it around the gel and supporting glass plate to avoid curling of the gel. Avoid trapping air. Let the wrapped gel dry in a well-ventilating oven at 50°C.

19.

Photograph the gel for a permanent record.

  Notes1. Prefocusing of the plates prior to sample application for establishing pH gradient is preferable.2. The sample may preferably be applied at the cathode and where the denaturation of most proteins

does not take place, although theoretically sample can be applied at any place, for focusing, however.3. Agarose is also used as a supporting medium for analytical IEF.4. Preformed gels for focusing are available commercially.5. Preparative electrofocusing is done using vertical column stabilized by density gradients with sucrose,

glycerol and ficoll.  References 1. Aweleh, Z L, Williamson, A R and Askonas, B A (1968) Nature  219 66.2. Wrigley, C W (1968) J Chromatogr 36 362.3. Radola, B J and Grasslin D (1977) In: Electrofocusing and Isotachophoresis (Ed de Gruyter) Berlin and New York.4. Ranjan Prasad, Chandrasekaran, P and Sadasivan, S (1986) Genet Agr 40 255.

Quantification of proteins in polyacrylamide gels

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is undoubtedly a versatile technique for the characterization of proteins both quantitatively and qualitatively. It is an easy and quick method to quantify particular proteins at microgram level from a mixture. This is done by scanning the gel (strips) and by densitometry of stained bands on it. Similarly, radioactivity-labeled polypeptides electrophoresed on SDS-PAGE can also be quantitated by densitometric scanning of the fluorographic plate.

PrincipleThe percent absorption of incident light is directly proportional to the color intensity of the protein-dye complex on the gel and is directly related to the protein concentration.Similarly, the intensity of darkening of the X-ray plate is directly proportional to the radioactivity in the protein in fluorographic plates.  Materials A spectrophotometer with suitable scanning facility and chart recorder (or integerator facility, if available) Protein Stain (Quantitative): 0.2% Proceion Navy MXRB dye in Methanol : Acetic Acid : Water (5:1:4). Dissolve the dye first in the methanol and then proceed. Prepare fresh everytime. Destaining Solution: Methanol : Acetic Acid : Water (1:1:8).For Fluorograph Scan Fluorograph Plate (see fluorography)  Procedure1. After the electrophoresis (see SDS-PAGE of protein), immerse the gel in Proceion Navy dye solution

and shake until the proteins are completely stained (for a fixed period say 2h).2. Destain the gel until the background is colorless.3. Scan the gel at 580nm to measure the degree of dye bound by each band of protein. Depending upon

the type of equipment available for scanning, the whole gel is used or each lane is cut out and scanned individually. The total absorption by the dye in each band is proportional to the area of the peak in the scan profile.Each peak in the scan profile is traced using a planimeter to determine the area under it. Otherwise, each peak in the chart may be cut out and weighed. When an integrator is interposed, the area under each peak is automatically calculated.

4. A curve is obtained by plotting A580 vs. amount of protein used as standard. Bovine serum albumin (Fraction V) at different known concentrations co-electrophoresed in different lanes in the same gel is also used to construct the standard curve. It should however be noted that the protein both in the standard and under examination to have equal dye-binding property.

Scanning Fluorographic Plate5. Scan the individual lane strip or the whole fluorographic plate at 620nm as described above. The

standard curve is obtaining using a radioactivity labeled standard protein whose concentration and radioactivity are known.

  

Notes1. The following conditions need to be satisfied to quantity proteins on the gels:

a) The protein bands should be well resolved, b) the dye should be bind to the protein of interest, and the binding should be uniform to all proteins and c) the sampling errors should be small

2. If the peaks are not well resolved, use of the narrower beam of light will improve the situation but at the cost of baseline.

3. Coomassie brilliant blue R250 staining is not suitable for quantitative analysis of proteins although it is a highly sensitive stain.

4. Proceion Navy  dye binds to the proteins stochiometrically and covalently. Destaining of this dye from the gel requires longer time.

5. Sampling errors are inevitable but their effect can be reduced by repetition and averaging the results.6. Electrophoresis with a fixed sample volume, voltage and duration of run is necessary between runs to

obtain satisfactory results.7. Gel scanner with computer facility are now commercially available.

  References 1. Carlier, A R, Manickam, A and Peumans, W J (1980) Planta 149 227.2. Smith, B J, Toogood, C and Johns, E W (1980) J Chromatogr 200 200.

Rocket Immunoelectrophoresis A number of methods are available for the quantitative estimation of a particular protein in a mixture. These include electrophoresis and immunochemical methods. The rocket immunoelectrophoresis is such a simple, rapid and reliable method, since rocket-like immunoprecipitate is formed when the desired protein (antigen) is electrophoresed in an agarose gel containing its monospecific antiserum. Since the height of the peak depends on the relative excess of antigen over antibody, a comparison of the peak heights of the unknown and standard samples allows the unknown protein concentration to be determined. This method was first described by Laurell in 1966. Although it is widely used in the clinical laboratories, quantitative estimation of seed proteins etc., could be easily carried out.  PrincipleWhen the protein sample (antigen) is electrophoresed into the agarose gel containing the monospecific antiserum in the presence of excess antigen, the Ag-Ab complex is soluble but as the antigen advances, more antigen molecules combine with antibody until and equilibrium is reached. At this stage, the Ag-Ab complex is insoluble, resulting in the precipitation spreading as a rocket from the antigen well. The area of the height of the rocket is then a measure of antigen concentration.  Materials 0.06M Barbitone Buffer             (pH 8.4)Sodium Barbitone                       10.3gBarbitone                                      1.84gWater                                             1L

Electrode Buffer is prepared by diluting it 1:1 with distilled water 2% Agarose (electrophoresis grade) in distilled water Antiserum to the protein under investigation. Staining Solution: 0.1% Coomassie brilliant blue R250 in 50% methanol & 10% acetic acid Destaining Solution: 10% Methanol 7% acetic acid Glass plates, size 5 x 5 x 0.1cm or 7.5 x 7.5 x 0.1cm Electrophoresis System Procedure1. Place a clean dry glass plate (5 x 5 x 0.1cm) on a leveled surface.2. Melt the 2% agarose either in a boiling water bath or in an autoclave. Transfer the flask to a water

bath at 52°C. stand a 5mL graduated, wide mouth pipette in this solution. Allow some minutes to cool the agarose solution in 52°C, and at the same time warm up the pipette.

3. Place a test tube in a water bath and add 2.8mL of 0.06M barbitone buffer to the test tube. Leave this for 3-5min to warm up. Then quickly transfer 2.8mL of agarose solution to this tube using the wide mouth pipette. Otherwise agarose will set in the pipette. Briefly mix the contents of the tube and return to the water bath.

4. Add 25-50mL of antiserum to the diluted agarose solution and briefly mix for even dispersion of the antiserum. Return to the water bath to allow any bubbles to settle out. (The appropriate volume of antiserum to be used depends on the antibody titre and has to be determined by trial and error).

5. Pour the contents of the tube slowly onto the glass plate keeping the neck of the tube close to the tube. Surface tension will prevent the liquid running off the edge of the plate. If necessary, tape can be used to form an edging to the plate. The gel will be approximately 2mm thick.

6. Allow the gel to set for 5-10min and then make holes using a Pasteur pipette attached to a water-pump vacuum at 0.5cm spacings 1cm in from one (cathode) side of the plate.

7. Place the gel plate on the cooling plate of the electrophoresis tank. Pour the electrode buffer into the tank compartments. Place the electrode into the tank compartments. Place the electrode wicks (five layers of Whatman No.1 prewetted in electrode buffer) over the edges of the gel. The wicks should not overlap the sample wells. The wells should be at the cathode end.

8. Deliver suitable aliquots (1-5L) of sample of each well using a microsyringe. The samples should include a few different concentrations of the pure protein (antigen) and a few dilutions of each unknown sample. Loading of the wells should be completed at the shortest duration or a small current (1-2mA) may be applied during loading in order to avoid any diffusion of the sample.

9. Immediately after loading, raise the current to 20mA and run for 2.5-3h. Gels can also be run at 2-3mA overnight. In any case, efficient cooling of the plate is essential.

10.

After the run, remove the gel plate from the apparatus. Precipitation rockets can be seen in the gel.

11.

Prior to staining, wash the gel with several changes in saline solution for 4-5 hours to completely remove the unreacted proteins. The gel may be stained wet or after drying to give a permanent record.

12.

The gel is dried by blotting. With the gel on a sheet of clean glass, place 6-10 sheets of Whatman No. 1 filter paper on top of the gel and apply heavy weight for 2-3h. Then the top wet filter papers are carefullyremoved to reveal the flattened and nearly dry gel. Further drying of the gel is continued with a stream of warm air using a hair-dryer.

13.

The wet or dried gel is now gently shaken in the staining solution for 15-20 min and subsequently destained.

14.

Measure the height (mm) of each rocket and plot a graph of peak height versus concentrations of standard.

15 The protein concentration in an unknown sample is calculated from the graph.

.  Notes1. Small aliquots of 2% agarose solution can be stored in separate tubes and each time a tube withdrawn fresh for use.2. It is essential to standardize the dilution of the antiserum and the concentration of the standard and sample proteins by trial and error runs for the right size of the rockets for comparison.  References 1. Laurell, C B (1966) Anal Biochem 15. 2. Walker, J M (1984) In: Methods in Molecular Biology Humana Press New Jersey Vol. 1 pp 317.

Protein (Western) Blotting The transfer of protein bands from an acrylamide gel onto a more stable and immobilizing support is called as protein blotting. A number of supporting matrices such as nitrocellulose, diazobenyloxymethyl cellulose sheets, nylon filters etc are used for the purpose. The separated proteins are buried in polyacrylamide gels and therefore further analysis of proteins or their recovery is cumbersome. However, the proteins can be efficiently transferred from the gel to the supporting medium by blotting. A variety of analysis involving immunoblotting, DNA-binding proteins, and glycoproteins could then be performed on the proteins blotted onto the filters. This method is an extension of the ‘Southern blot’ used to transfer DNA from gels to nitrocellulose filter and is called as the ‘Western blotting’. The beneficial of a protein blot include rapid staining/destaining detectio9n of proteins at low concentrations and rapid localization of the proteins in preparative gels. The blots can be preserved as replica of the original gels. The transfer of proteins is carried out either by electrophoresis (electroblotting) or by the capillary action of buffer (capillary blotting). The latter method is described below.  PrincipleThe separated proteins are transported out of the gel either by the capillary action of the buffer or in an electric field. The presence of SDS increases the solubility of proteins and thus facilitates the migration of proteins. Once out of the gel, the proteins comes in contact with the nitrocellulose membrane which binds the protein very strongly onto the surface as a thin band thus producing a replica of original gel.  Materials Nitrocellulose Paper (pore size 0.20-0.45m) Blotting Buffer (pH 8.3)0.02M Tris-HCl           2.42g

0.15MGlycine             10.25g20% Methanol            200mLWater to                       1L(can be stored at 4°C for 2-3weeks). Protein Stain: 0.01% Amido black 10B in Methanol:Acetic acid:Water (5:1:5) Destaining Solution: Methanol:Acetic acid:Water (5:1:5)  Procedure1. Arrange a platform by placing a 25 x 20cm glass plate at a suitable height on the work bench.2. Assemble six layers of Whatman No. 1 filter paper and place over the platform. Dip the short ends of

the papers in two glass trays containing the blotting buffer placed on either side of the platform. Let the papers wet thoroughly. The papers should be wide enough to accommodate the gel to be blotted.

3. After the separation of proteins in slab gels by SDS-PAGE, discard the stacking gel and carefully lay the separation gel to be blotted on the wetted filter paper. Cut the required portion of the gel with a scalpel blade if the whole gel is not to be blotted, and lay carefully.

4. Take a piece of nitrocellulose paper exactly the same size of the gel to be blotted. Wet thoroughly by floating on the blotting buffer. Carefully place this wetted nitrocellulose paper over the gel without trapping any air bubbles. This is conveniently done by lowering first the middle of the paper on the gel and then laying towards the ends. It is again preferable to wet the top of the gel with blotting buffer before layering the nitrocellulose paper.

5. Cut out the middle of a cling film to the size of the gel, and lay it over the nitrocellulose paper such that the film does not cover the nitrocellulose paper. In other words, the cling film will form a frame revealing the nitrocellulose paper covering the surrounding portions. This shall prevent by-pass of buffer from the bottom layers to top layers of filter papers directly. This is to ensure on any account the buffer should pass only through the gel.

6. On top of the film, lay six layers of Whatman 3mm filter paper cut to the same size as the gel followed by a way of absorbant material (tissue paper or disposable nappy) also of the same size of the gel.

7. Place a heavy weight for example a conical flask containing one liter water, over the sandwich and leave the set up for 1-2 days. There should be ample blotting buffer in the tanks during blotting.

8. The buffer from the bottom layer of filter papers moves upward by absorption via the gel and nitrocellulose paper. During this process, the proteins are transferred by capillary action from the gel to the nitrocellulose which has more attraction to the protein.

9. After blotting for required period, recover the nitrocellulose paper disassembling the set-up. The nitrocellulose paper can be stored pressed between a fold of filter papers until required for further analysis or can be stained directly.

10.

Immerse the protein blot in the amido black dye solution for 10-15min with gentle shaking. Subsequently, destain the blot in the destaining solution with repeated changes. The transferred proteins are visualized as black bands. The blot is then dried between several sheets of filter paper held flat with a heavy weight.

  Notes1.

The nitrocellulose sheets should be stored air tight at 4°C to prevent them being contaminated by volatile chemicals etc.

2.

While assembling the sandwich for blotting care must be taken to see that there is no direct contact of nitrocellulose paper or the above layers with the bottom layers of filter paper in order to avoid the flow of the buffer directly. It should only flow through the gel to the nitrocellulose paper in an ideal blot assembly.

3.

Nitrocellulose paper should be thoroughly wetted before being used.  Any bubbles between the gel and nitrocellulose paper will result in poor transfer of proteins.

4.

Capillary blotting is a passive process and requires longer period (2-4 days). The duration of blotting largely depends upon various factors such as the porosity of nitrocellulose, percentage of acrylamide and thickness of the gel, the ionic strength of the blotting buffer, the solubility of proteins etc. the low MW proteins are easily transferred than the high MW proteins.

5.

The transfer of proteins by electroblotting is much faster and efficient than the capillary blotting method. Suitable cassettes to assemble the sandwich for electro-blotting are commercially available.

6.

Equilibrium of the gel prior to blotting for renaturing of the separated proteins is suggested depending upon the further analyses of the blot. The gel is equilibrated in the following buffer with constant shaking for 30-60min prior to capillary blotting for renaturing the separated proteins.1M Tris-HCl pH 7.0              5mL5M NaCl                                 5mL0.1M EDTANa2                      10mL0.1M Dithiothreitol                                0.5mLUrea                                        120.12gWater to                                  350mL

7.

Wear gloves while handling the gel and nitrocellulose paper to avoid contamination. Laying a second nitrocellulose paper at the bottom of the gel is also a common practice in contact-diffusion blotting. By this way two blots are obtained simultaneously.

  References 1. Towbin, H, Staehelin, T and Gordon, J (1979) Proc Natl Acad Sci USA 76 4350.2. Burnetter, W N (1981) Anal Biochem 112 195.3. Manual on ‘Techniques in Molecular Biology’ (Proteins) (1986) Workshop held at the Department of Biochemistry Tamil Nadu Agricultural University Coimbatore p 51.

Silver staining of proteins The fractionated polypeptides on gels are visualized, by staining generally either with coomassie brilliant blue R 250 or amido black 10B dye. The above dyes can detect a band containing a little as 0.1g of polypeptide. In may occasions, the available protein for electrophoresis is so small or some proteins occur in minute amounts the detection becomes extremely difficult with these dyes. Under such circumstances a higher sensitive detection system is required. Silver staining is a very useful method in this regard with about 100-fold greater sensitivity over dye staining. It is comparable in sensitivity to autoradiography of labeled polypeptides. There are different methods described by different workers for silver staining. The method given below is very simple and rapid.  PrincipleThe amino acids particularly aromatic amino acids in the protein reduce silver nitrate and form complexes with metallic silver of yellowish-brown to brown color.   Materials Washing solutionMix 1mL of formaldehyde (analytical grade, 37%), 40mL of methanol and 60mL of distilled

water and use. Sodium thiosulphate: dissolve 200mg in a liter of water Silver nitrate solution (0.1%) Developer: dissolve sodium carbonate 3g (w/v) in about 80mL water. Add 1mL of the above sodium thiosulpahte solution and 1mL of formaldehyde and finally make up the volume to 100mL with water. Stopper: 5% citric acid or 5% acetic acid solution.  Procedure1. After electrophoresis, transfer the gel to a clean plastic container and wash the gel in the washing

solution with slow shaking for 10min.2. Discard the wash solution and rinse the gel with plenty of water for 2min.3. Soak the gel in sodium thiosulphate solution for 1-2min.4. Wash the gel with water twice, each time 1-2min. drain the wash water.5. Soak the gel in silver nitrate solution for 10min with gentle shaking.6. Wash the gel in water as in step 4.7. Pour developer to the plastic container and shake the gel slowly, gently. The proteins reduce silver

nitrate to silver and the yellow to dark brown color bands appear.8. When sufficient intensity of band developed stop the reaction by adding either citric acid or acetic

acid solution.9. Record the protein banding pattern by photography.

  Notes1. Cleanliness is very much important in this experiment since the reaction is very sensitive. Use filtered

reagents. All glassware should be thoroughly cleaned.2. Wear gloves while working, particularly when handling the gel; otherwise fingerprints will be a

problem.3. The gel becomes very fragile during the treatment, exercise adequate care.4. The method is very rapid and easy. The proteins can be fixed in the gel, if necessary, by immersing it

in 10% glutaraldehyde for 30-60min and washed in water prior to washing the gel in washing solution mentioned above.

5. Each washing step should be done effectively.6. Continuous gentle shaking of the gel in every step improves uniform staining.7. Destained and/or unstained gels can be used for detection of proteins. Destained gels are more rigid

and can be handled easily than the unstained gels.8. The color formation is inorganic in reaction and hence proceeds very rapidly. Stop the reaction once

the required intensity of bands is obtained by pouring stopper solution and with washing.  References 1. Oakley, B, Kirsch, D and Morris, N R (1980) Anal Biochem 105, 361.2. Merril, C R, Danan, M and Goldman, D (1981) Anal Biochem 110, 201.3. Poehling, H M and Neuhoff, V (1981) Electrophoresis 2, 141. 

Protein digestibility in vitro 

Protein quality determination is important in the production of nutritionally improved varieties of cereals and pulses. The nutritive value of a protein depends primarily on its capacity to supply needs of nitrogen and essential amino acids. Although the chemically determined amino acid composition is used to measure the quality of a protein, the biological availability of these amino acids is the real measure of the quality of the proteins. The availability of amino acids depends on the extent of digestibility of proteins by the proteolytic enzymes of the alimentary tract. Digestibility of a protein can be assessed using rats which is termed in vivo digestibility. It can also be measured using proteolytic enzymes and called in vitro protein digestibility. The results obtained with the later procedure agreed well with in vivo protein digestibility as measured in the rats. The procedure described here is that of Hsu et al1 which was later modified by Satterlee et al2.  PrincipleFour proteolytic enzymes are used to digest the protein and the pH changed due to the release of amino acids at a fixed time interval is measured. By using the formula - % digestibility = 234.84 – 22.56 X, where X is the pH after 20min incubation, the in vitro digestibility is arrived at.  Materials Powdered sample which passes through 80-mesh screen Glass distilled water Three enzyme solution: 1.6mg trypsin, 3.1mg chymotrypsin and 1.3mg peptidase per mL in glass distilled water. Bacterial protease solution: 7.95mg protease (type IV from Streptomyces griseus) per mL in glass distilled water.  Procedure1.

Add 10mL of glass distilled water to the powdered sample (amount of sample is adjusted so as to contain 6.25mg protein/mL).

2.

Allow the sample to hydrate for at least 1h (not longer than 25h) at 5°C.

3.

a) Equilibrate the sample to pH 8.0 at 37°C.b) Equilibrate the three enzyme solution to pH 8.0 at 37°C.

4.

Add 1mL of three enzyme solution to the sample suspension and stir while being held at 37°C.

5.

After exactly 10min from the time of addition of three enzyme solution (still stirring) add 1mL of the bacterial protease solution.

6.

Immediately transfer the solution to 55°C water bath.

7.

Nine minutes after adding the bacterial enzyme, transfer the solution back to 37°C water bath (in total 19min after the addition of the three enzyme solution).

8.

Measure the pH of the hydrolysate exactly 10min after the addition of bacterial enzyme. This is called the 20min pH.

  CalculationIn vitro protein digestibility is calculated using the following equation:- % digestibility = 234.84 – 22.56 Xwhere X is the pH after 20min incubation  NoteFirst run a control (HNRC sodium caseinate) before each set of samples and this must have a 20min pH of 6.42 ±0.05. this control is needed to ensure the presence of proper enzyme activity prior to any running samples.

References 1. Hsu, H W, Sutton, N E, Banjo, M O, Satterlee, L D and Kendrick, J G (1978) Fd Technol 32 69.2. Satterlee, L D, Marshall, H F and Tennyson, J M (1979) J Am Oil Chem Soc 56 103.

Amino Acid Analysis:

Introduction

In many cases an exact knowledge of protein quantities is required for further protein chemistry applications. Amino Acid Analysis is the suitable tool for precise determination of protein quantities, but also provides detailed information regarding the relative amino acid composition and free amino acids. The relative amino acid composition gives a characteristic profile for proteins, which is often sufficient for identification of a protein. It is often used as decision support for choice of proteases for protein fragmentation.

The procedure includes:

o Hydrolysis o Separation, Detection and Quantification

Hydrolysis

Hydrolysis is typically achieved by acid conditions. A standard procedure is hydrolysis with 6 M hydrochloric acid (24 hours, 110°C). These standard procedure is a compromise between time requirement and temperature. Sensitive amino acids (especially tryptophane and cysteine) will be partially destroyed. Gas phase hydrolysis and addition of other acids (e.g.

propionic acid, TFA, methansulfonic acid) can be used to shorten the hydrolysis time and improve the yield of sensitive amino acids.

Separation, detection and quantification

Hydrolysed samples(amino acids)are derivatized for sensitive detection,separated by HPLC. Whereas post-column derivatization was typical earlier, pre-column derivatization has gained importance and can be achieved by a broader range of derivatization reagents. The use of internal and external standards is crucial.

4.4. D amino AcidsThe D-amino acid content of a protein or peptide can be determined by employing a short partial acid hydrolysis, followed by an enzymatic hydrolysis with pronase, and then with leucine aminopeptidase and peptidyl-D-amino acid hydrolase (25).