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2. Principal:
o 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.
o A polypeptide chain binds amounts of SDS in proportion
to its relative molecular mass.
o 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.
3. o Poly acrylamide gels restrain larger molecules from migrating
as fast as smaller molecules.
o 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.
4.
5. Re
a
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 microliter)
o 1M Tris HCL (312.5microliter)
o 10% SDS (25micro-liter)
o 10% APS (25 micro-liter)
o TEMED (3micro-liter)
6. o Wash glass plates and spacers in warm detergent solution
and rinse with tap water deionized water.
o Rinse plates with ethanol and dry it. The glass plates musd be
free of grease spots to prevent air bubbles in gel.
o Assemble the glass plates with spacers.
o Prepare Gel solution with desired poly acrylamide percentage,
which gives amount of each component required to make
100ml.
o Add 35 micro-liter of TEMED for each 100ml of acrylamide,
bis solution, and mix the solution with gentle swirling.
o Gels can be cast with 1 micro-liter TEMED per ml of gel
solution to increase rate of polymerization.
o Immediately insert comb into gel, being careful not to allow
air bubbles become trapped under teeth.
o Allow acrylamide to polymerize for 30-60 minutes at room
temperature.
7. o After polymerization is complete, surround comb and top of
gel with paper towels soaked in 1X TBE.
o Seal the entire gel and store it at 4’C untill needed.
o When ready to proceed for electrophoresis Squirt 1X TBE
buffer, pull comb form polymerized gel.
o Use syringe to rinse out wells with 1X TBE, remove tape.
o Attach gel to electrophoresis tank, using clips on sides.
o Fill reservoirs of electrophoresis tank with TBE buffer.
o Use syringe to flush out wells once more with 1X TBE. Mix the
DNA samples with appropriate amount of 6X-Gel loading
buffer.
o Load the mixture into wells using micro-pipette.
o Connect electrodes with power pack (positive electrode
connect to bottom reservoir), turn on the power and begin
the electrophoresis run.
8. o Run the gel untill the marker dyes have migrated the desired
distance.
o Turn off electric power, disconnect the leads, and discard the
electrophoresis buffer from the reservoirs.
o Detect the positions of bands of DNA in the polyacrylamide
gel.
9.
10. o Page has high loading capacity, up to 10
micrograms of DNA can be loaded into single
well without significant loss of resolution.
o Page is an ideal system from which isolate
DNA fragments for sub-cloning and other
molecular biological techniques.
11. o Separate from other proteins on the basis of
molecular weight and size.
o Determine molecular size of protein.
o Determine quantifies amount present.
o Used in western blot assay.
