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SDS-PAGE Principle
Electrophoresis is the study of the movement of charged molecules in an electric field. The generally used support medium is cellulose or thin gels made up of either polyacrylamide or agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger molecules like proteins. The general electrophoresis techniques cannot be used to measure the molecular weight of the biological molecules because the mobility of a substance in the gel is influenced by both charge and size. In order to overcome this, if the biological samples are treated so that they have a uniform charge, electrophoretic mobility then depends primarily on size. The molecular weight of protein maybe estimated if they are subjected to electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing agent mercaptoethanol ( ME). SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein.
Chemical Polymerization
Polyacrylamide
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Pull the completed sandwich from the alignment slot. Check that the plates and spacers are aligned. If not, realign the sandwich as in steps 1 Before transferring the clamp assembly to the casting slot, -3. recheck the alignment of the spacers. Do this by inverting the gel sandwich and looking at the surface of the 2 glass plates and the spacer. Make sure that they are aligned. Transfer the clamp assembly to one of the casting slots in the casting stand. If 2 gels are to be prepared, place the clamp assembly on the other side of the alignment slot. Press the acrylic pressure plate bottom, so that the glass plates rest on the rubber gasket. Snap the acrylic plate underneath the overhang of the casting slot. Do not push the glass plates or spacers because this could break the glass plate. Figure 1
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Add APS and TEMED to the solution and pipette the solution down one of the spacer until the sandwich is filled completely. Allow the gel to polymerize for 15 minutes. Remove the comb. Gel is placed in the buffer chamber and running gel buffer is added into the chamber
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Sample buffer
(Glycerol, SDS, Mercaptoethanol, Bromophenol blue dye)
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20l
Do not boil
5 min 20l
5 min 20l
5 min 20l
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Appendix 1 Preparation of a 10% Resolving/separating gel. 30% acrylamide + 0.8% Bis-acrylamide 4X Tris-HCl/SDS, pH 8.8 Distilled water 10% ammonium persulfate (APS) TEMED 1.25 ml 0.94 ml 1.56 ml 25 l 10 l
Preparation of a 4.5% stacking gel 30% Acrylamide + 0.8% Bis-acrylamide 4X Tris-HCl/SDS, pH 6.8 Distilled water Ammonium persulfate TEMED 0.65 ml 1.25 ml 3.05 ml 25 l 5 l