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Methodology for Amino Acids and Proteins

 
     
 
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.
  SDS Page
 
SDS-PAGE

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.
 


 
Principle
SDS 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 Solution
Acrylamide 30%                                                         30g
Bisacrylamide 0.8%                                                    0.8g
Water to                                                                     100mL
 
Separation Gel Buffer
1.875M Tris-HCl                                                          22.7g   pH 8.8
Water to                                                                    100mL
 
Stacking Gel Buffer
0.6M Tris-HCl7.                                                            26g   pH 6.8
Water to                                                                     100mL
 
Polymerizing Agents
a) Ammonium persulphate 5%                                0.5g/10mL, prepare freshly before use
b) TEMED                                                                 fresh from the refrigerator
 
Electrode Buffer
0.05M Tris                                 12g
0.192M Glycine                          28.8g   pH 8.2-8.4
0.1% SDS                                  2g No adjustment required
Water to                                    2mLmay be used 2-3 times
 
Sample Buffer (5X concentration)
Tris-HCl buffer pH 6.8                 5mL
SDS0.                                         5g
Sucrose                                      5g
Mercaptoethanol0.                     25mL
Bromophenol Blue                      1mL
(0.5% W/V solution in water)
Water to                                    10mL
Store frozen in small aliquots. Dilute to 1x concentration and use.
 
Sodium dodecyl sulphate 10% solution – store at room temperature.
 
Standard Marker Proteins
Protein                                                                       MW Daltons
a-Lactalbumin                                                           14,200
Trypsin inhibitor soyabean                                        20,100
Trypsinogen                                                              24,000
Carbonic anhydrase                                                  29,000
Glyceraldehyde-3-phosphate dehydrogenase,
rabbit                                                                        36,000
Albumin, egg                                                             45,000
Albumin bovine                                                         66,000
Dissolve the above proteins in single strength sample buffer at a concentration each of 1mg per mL. Load the well with 25-50mL.
 
Protein Stain Solution
Coomassie brilliant blue R 250                                0.1g
Methanol                                                                40mL
Acetic acid                                                              10mL
Water                                                                     50mL
First, dissolve the dye in methanol and proceed. Use fresh preparation every time.
 
Destainer
As above without the dye.
 
 


Procedure
1.
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% gel
Stock acrylamide solution
20mL
13.3mL
Tris-HCl (pH 8.8)
8mL
8mL
Water
11.4mL
18.1mL
Degas on a water pump for 3 – 5min and then add:
Ammonium persulphate solution
0.2mL
0.2mL
10% SDS
0.4mL
0.4mL
TEMED
20mL
20mL
 
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 = 1.35mL
Tris-HCl (pH 6.8) = 1mL
Water = 7.5mL
Degas as above, and then add:
Ammonium persulphate solution (5%) = 50mL
10% SDS = 0.1mL
TEMED = 10mL
Remove 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 (50-200mg) in a volume (25-50mL) 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’).
 
 
 
Notes
1.
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
Remedy
a.
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 gel
f.
Protein bands are wavy
Excess persulphate
Use optimum concentration of presulphate
 
Bands 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 0.1mg protein is visualized by coomassie brilliant blue staining in SDS-PAGE; for visualizing proteins of lower concentration below 0.1mg high sensitive (silver staining) method is prepared.
 
 
References
1. 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.

 
     
 
 
     




     
 
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