10. Sodium Dodecyl Sulphate Polyacrylamide gel Electrophoresis

( SDS - Page )



A. Principle :


SDS-PAGE, first described by Shapiro et al in 1967 and Weber and Osborn in 1969 is one of the most widely used techniques for the analysis of proteins based on their molecular weights. The binding of SDS to proteins under reducing conditions has two important consequences. The first is that it completely swamps the native charge of the protein, so that the charge: mass ratio becomes constant for all proteins. Secondly, the electrostatic repulsion between the negatively charged SDS molecules effectively disrupts virtually all non-covalent protein interactions, both intra-molecular and inter-molecular. The proteins may assume the shape of a rigid rod with a constant charge: mass ratio. Under these conditions it has been observed that the electrophoretic mobility of the protein in polyacrylamide gel is inversely proportional to the logarithm of its molecular weight. Proteins bind approximately 1.4g SDS per gram of protein. By comparing the electrophoretic mobility of a given protein with the mobility of appropriate standard proteins, its molecular weight can be determined. Polyacrylamide gel electrophoresis may be carried out in glass tubes or as slabs between two glass sheets. In general, the slab gel system is preferred because it allows multiple samples to be analysed in one gel and it also allows more precise alignment of different samples.




B. Materials:


a.Slab gel electrophoretic apparatus-complete system.


b.Acrylamide - 30% w/v


Acrylamide -     14.6 g


N, N Bisacrylamide                                       -     0.4 g


Dissolve in 30 ml double distilled water and adjust the volume to 50 ml. Filter through Whatman-1 filter paper and store at 40C in a brown bottle.




c. SDS - 10%


SDS     -                                                           2 g


Dissolve and adjust the volume to 20 ml in double distilled water.




d.  Upper tris (pH 6.8)


Tris base                                                           -     3.03 g


10% SDS                                                          -     2 ml


Dissolve in 30 ml double distilled water, adjust the pH to 6.8 with conc. HCl and make the final volume to 50 ml.




e.Lower tris (pH 8.8)


Tris base                                                           -     18.15 g


10% SDS                                                          -     4.0 ml


Dissolve in 80 ml double distilled water, adjust the pH to 8.8 with conc. HCl and make  the final volume to 100 ml.


f. SDS sample buffer


Upper tris                                                         -     1.25 ml


10% SDS                                                          -     3.0 ml


Glycerol                                                            -     1.0 ml


Double distilled water                                        -     4.75 ml


Bromophenol blue                                              -     10 mg


(for reduction add 500 µl of mercaptoethanol).


Mix together and store at 40C in a glass vial.




g.Running buffer.


Tris -     3.03 g


Glycine                                                           -     14.40g


SDS -     1g


Make volume to 1 litre with double distilled water.


h. Ammonium persulphate solution


Dissolve 0.1 g ammonium persulphate in 900 µl double distilled water.


i.Bm mf ' s ' antigen    (Appendix-II)


j.Standard molecular weight markers   (Appendix-III)




C. Method :

1.The  slab  gel  is prepared  between two glass plates. Clean the glass plates first with a detergent and then thoroughly with distilled water.


2.Place  the  two glass plates together, with the spacers between them ( along  the  edges )  and fix the whole assembly tightly with clamps or gel casting stand.


3.Seal  the  two  side edges  and  the bottom  edge  with 2% agar and keep the assembly vertically on a flat surface.


4.Prepare separating gel according to the protocol given here.


Lower tris                                                         - 9.4 ml


Double distilled water                                     -     15.6 ml


Acrylamide                                                       -     12.6 ml


Ammonium Persulphate                                  -     150 µl


TEMED                                                              -     22 µl


Mix solutions without TEMED in a 100 ml conical flask and deaerate.


5.mix and pour immediately between the glass plates, upto about 2.5 cm


below the notch.


6. Overlay the gel with water saturated n-butanol in order to achieve an even surface.  The polymerization   is completed in 30-60 minutes at room temperature


7.Pour out the upper water layer and unpolymerized gel. Rinse with water twice to ensure the removal of unpolymerized gel.


8.Prepare stacking gel in a small beaker according to the protocol given here.


Upper tris                                                         -     4.0 ml


Double distilled water                                     -     9.6 ml


Acrylamide                                                       -     2.4 ml


Ammonium persulphate                                 -     300 ml


TEMED                                                            -     16 ml


Deaerate the solution before adding TEMED.




9.Pour stacking gel and insert comb into the stacking gel (Avoid trapping of air bubbles.)


10.Allow the stacking gel to polymerize for 15 to 20 minutes.


11.Carefully remove the comb by sliding vertically upwards.


12.Rinse the slots with distilled water 3 to 4 times to washout the unpolymerized gel.


13.Remove the clamps, the bottom spacer and clean the bottom edge with a tissue paper. Fix the gel assembly to the electophoresis chamber with the help of clamps (with notched plate inside).


14.The slab gel plates are in air tight contact with the electrophoretic chamber because of the rubber gasket. Seal the rubber gasket with 2% agar to prevent any possible leakage of buffer from the upper chamber.


15.Fill the upper and the lower chambers  with running buffer. Remove the air bubbles trapped at the bottom edge of the gel between the glass plates using a  syringe  with a bent needle or by tilting to one side. Connect the electrodes to the power supply and make pre run at a constant current of 20 mA for 15 minutes.


16. Switch off the power supply, apply samples  ( 5 to 50 ml )  and molecular  weight standards (10 ml) to the slots with the help of a micro syringe or micropipette.


17.Switch on the power supply and run electrophoresis at a constant current of 20 mA till the samples reach separating gel and then increase current to 25 mA.


18. Run electrophoresis till the tracking dye reaches the end of the separating gel.


19. After  the completion  of  run, disconnect  the  power supply,  remove the gel assembly from  the  electrophoresis chamber  and  then remove the side spacers and the notched glass plates with the help of spatula.


20. Now the gel may be stained or used in blotting experiment.


Note :


For SDS-PAGE on mini gel (8.5 x 7.5 cm) reduce the volume of the solutions to half while preparing running gel & stacking gel.


References :


1. Shapiro, A. L., Vinuela, E. and Maizel, J. V. Molecular weight estimation of polypeptide chains  by  electrophoresis  in  SDS  - polyacrylamide  gels,  Biochem. biophys.  Res. Commun., 28 (1967), 815.


2.Weber, K. and Osborn, M. The reliability of molecular weight determinations by dodecyl sulphate polyacrylamide gel electrophoresis. J. Biol. Chem., 244 (1969) 4406.