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.
5.
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.
10.
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.
12.
13.
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.