The document describes SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique used to separate proteins based on their molecular weight. SDS denatures proteins and gives them a uniform negative charge, allowing separation based primarily on size in the polyacrylamide gel. The document provides details on preparing the resolving and stacking gels, running the electrophoresis, and using SDS-PAGE to separate and analyze proteins.

1. M.PHOOL BADSHAH

3.  Principal:
 SDS (also called sodium lauryl sulphate) is an anionic detergent meaning
that when dissolved its molecules have a net negative charge within a wide
PH range.
 A polypeptide chain binds amounts of SDS in proportion to its relative
molecular mass.
 The negative charges on SDS destroy most of the complex structure of
proteins, and strongly attracted toward an anode (positive charged
electrode) in an electric field.

4.  Polyacryalamide gels restrain larger molecules from migrating as fast as
smaller molecules.
 Because the charge-to-mass ratio is nearly the same among SDS-denatured
the polypeptides, the final separation of proteins is dependent almost
entirely on the differences in relative molecular mass of polypeptides.

6.  Resolving Gel (7.5 ml)
o Water (2.475ml)
o Acrylamide (3ml)
o 1.5 M Tris HCL (1.875ml)
o 10% SDS (75micro-liter)
o 10% APS (75microliter)
o TEMED (3micro-liter)
 Stacking Gel (2.5ml)
o Water (1.720ml)
o Acrylamide (495 micro liter)
o 1M Tris HCL (312.5microliter)
o 10% SDS (25micro-liter)
o 10% APS (25 micro-liter)
o TEMED (3micro-liter)

7.  Wash glass plates and spacers in warm detergent solution and rinse
with tap water & then deionized water.
 Rinse plates with ethanol and dry it. The glass plates must be free of
grease spots to prevent air bubbles in gel.
 Assemble the glass plates with spacers.
 Prepare Gel solution with desired Polyacryalamide percentage, which
gives amount of each component required to make 100ml.
 Add TEMED for each 100ml of Acrylamide, bis solution, and mix the
solution with gentle swirling.

8.  Gels can be cast with 1 micro-liter TEMED per ml of gel solution to
increase rate of polymerization.
 Immediately insert comb into gel, being careful not to allow air bubbles
become trapped under teeth.
 Allow Acrylamide to polymerize for 30-60 minutes at room
temperature.
 After polymerization is complete, surround comb and top of gel with
paper towels soaked in 1X TBE.
 Seal the entire gel and store it at 4’C until needed.
 When ready to proceed for electrophoresis Squirt 1X TBE buffer, pull
comb form polymerized gel.

9.  Use syringe to rinse out wells with 1X TBE, remove tape.
 Attach gel to electrophoresis tank, using clips on sides.
 Fill reservoirs of electrophoresis tank with TBE buffer.
 Use syringe to flush out wells once more with 1X TBE. Mix the DNA
samples with appropriate amount of 6X-Gel loading buffer.
 Load the mixture into wells using micro-pipette.
 Connect electrodes with power pack (positive electrode connect to
bottom reservoir), turn on the power and begin the electrophoresis run.
 Run the gel until the marker dyes have migrated the desired distance.
 Turn off electric power, disconnect the leads, and discard the
electrophoresis buffer from the reservoirs.
 Detect the positions of bands of DNA in the Polyacryalamide gel.

11.  Separate from other proteins on the basis of molecular
weight and size.
 Determine molecular size of protein.
 Determine quantifies amount present.
 Used in western blot assay.

14.  Page has high loading capacity, upto 10 micrograms of DNA
can be loaded into single well without significant loss of
resolution.
 Page is an ideal system from which isolate DNA fragemnts for
sub-cloning and other molecular biological techniques.