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Introduction
The separation of proteins by polyacrylamide gel electrophoresis (PAGE) is an important and
widely used biochemical technique. Used under denaturing conditions, specifically in the
presence of the anionic detergent sodium dodecylsulphate (SDS), it permits separation at very
high resolution of polypeptides from one another on the basis of their relative molecular
masses (the smallest polypeptides migrate fastest) and allows estimates to be made of their
relative molecular masses.
Note : TEMED and APS can be increased for faster polymerisation. Resolving gels may be
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stored for up to 1 week overlaid with storage buffer and water saturated n-butanol
Procedure
Note: Pour off overlay and rinse with water before pouring stacking gel
• Mix the stacking gel solution by swirling together. Pour on top of resolving gel until
the level is almost full and insert a Teflon comb, being careful not to introduce and trap
air bubbles.
• Leave until the gel has set. Allow 15-30 minutes for polymerisation.
• Once the stacking gel has polymerised the gel should be used within 60 minutes.
• Fit the cassette into the base reservoir, put a well guide onto the front so that you will be
able to see where the wells are and fill the back reservoir to the top with tank buffer.
Carefully remove the comb. Fill the base reservoir with tank/running buffer until it covers
the platinum wire. Make sure that the sample wells in the top of the gel are neat,
unclogged and visible.
• Using a Gilson P20 and the special long-nosed gel loading tips, load 30µl of each sample
and standards into the bottom of appropriate wells. Fit the cover and insert the leads into
the power pack (UNPLUGGED FROM THE MAINS!) so that the red is +ve and the
black is -ve. If the power pack has a polarity switch, check that it is set to POLARITY
NORMAL.
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• Make note of your loading scheme, keeping in mind the position of the molecular weight
marker which should be loaded asymmetrically, so that you may identify the lanes after
staining.
• Turn on the power, select CONSTANT VOLTAGE and adjust to 200V (or current to 20
mA) per gel.
• Run gel at 200 V for about 45 minutes. Leave until the blue tracker dye is below the top
of the buffer in the base. Turn off, unplug and strip down the cassette, discarding the
buffer down the sink.
• Carefully dismantle the cassette by SLIDING the spacers out of the top or bottom - DO
NOT PULL THEM SIDEWAYS. Use a thin spatula or razor blade to GENTLY lever the
glass plates apart AT THE SIDE OR BOTTOM CORNER – NOT AT THE TOP LUGS.
Cut off the bottom left corner of the gel. Transfer the gel to a polythene box. Please make
sure that your gel box is labelled.
• After removal of gel from cassette, separate portions of the gel to be used in blotting and
Coomassie staining by gently cutting it with a razor blade. Stain one part with Coomassie
blue. The other part will be retained for Western blotting.
Coomassie stain solution, for 1 L
• 2.50 g Coomassie Brilliant blue R-250 (CBB) (not blue G)
• 100 ml glacial acetic acid
• 400 ml methanol
• fill to 1 L with distilled water
De-stain solution, for 1 L
• 100 ml glacial acetic acid
• 200 ml methanol
• fill to 1 L with distilled water
Coomassie stain
• Allow that part of the gel to stain for an hour at room temperature. The destaining
will be continued for you for an additional few hours until an appropriate signal to noise
ration is obtained. This is when protein bands are clearly visible against a relatively
transparent background.
• Proceed to mass spectrometry preparation.
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composition of proteins and the molecular weights of their constituent polypeptides.
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9. After the excess liquid has run off the blot place it face down on the plastic
wrap and fold the wrap over enclosing the blot thoroughly so no residual liquid can
leak out. Ensure that the plastic wrapping is done smoothly with minimal creasing.
10. develop the film in one of the department’s automated developers