I was to carry out a research in our lab and the first step is to purify protein extracted from the sample and check bands using SDS-PAGE. The procedure used for the resolving gel are as follows; dH20-18.7ml, 30% Acrylamide, 0.8% Bis, 1.5M Tris, pH 8.8/0.4%SDS, TEMED-7.5ml, 10%APS-150ml and did not polymerize/rate of polymerization was extremely slow. What could i have gotten wrong?
Did you make fresh APS? (I'm assuming ml = ul for APS/TEMED here, incidentally)
APS goes off pretty quickly, so it's always worth making a fresh 10% stock.
Like what John said, APS is usually the main reason why your gel does not polymerise. Have you checked that your APS is dry? Unless it is stored in a desiccator, it slowly attracts moisture. If you can't find a fresh dry bottle of APS, try doubling the amount you add. Do you mean that you added 150 ul of 10% APS? I usually add 150 ul of 10% APS to a 10 ml mixture. If you mixture is 25 ml, I would add at least 300 ul.
Ideal time is, when you add stacking gel on the resolving gel, the resolving gel should have polymerize. that you can examine just by observing remaining content / leftover of the resolving gel. Always add APS at the end of all additions, swirl it and add immediately between plates.
Always prepare fresh APS and compulsorily air tight the lid of stock APS supported by parafilm.
This will solve problems associated with APS.
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