hello, when i pour alcohol (100% ethanol or iso-propanol) to overlay the resolving gel level, it penetrates the resolving gel, and makes a wavy line, or a trail that seems to pass across the resolving gel, is there any solution to this problem? I use 1.5 M tris , 30% bis acrylamide, 10%sds solution, 10% APS and Temed. looking forward for you guy's suggestions, TIA.
Is this supposed to be a Lämmli SDS-PAGE gel ? 1.5 M Tris sounds like it is the 4 x buffer. Otherwise, unless you are running non-denaturing gels, you can use 0.1 % SDS in water for the overlay. Just add it gently from the side, leaning the pipette tip against the spacer.
Navine, get a small (25 - 100 ml) glass bottle. Fill half way with iso-butanol (immiscible with water). Pipette in 1-2 ml of MilliQ-grade water. Shake. Let settle. If you dont see a small amount of water in the bottom, add a bit more water and repeat. Once you see a small layer of water in the bottom, your iso-butanol is saturated with water. Use this as your overlay solution. You only need to overlay with 100-400 ul (depending on mini vs maxi gel cell, gel thickness). When ready to add stack, pour off overlay. rinse surface of gel with MilliQ-grade water (from squeeze bottle or directly from water system), place gel on its side on the bench, use small strip of filter paper to remove remaining water (don't touch the gel with it, it will stick), place upright, add stack solution and comb.
Mix isopropanol and milliQ water. Shake and allow it to settle but make sure there is small amount of water at the bottom of the mixture. I think this will be ideal as said by Bruce Allen
Hi! Is there a particular way to remove isobutane after the resolving gel finishes polymerizing?
Tip the gel a bit, aspirate the iso, rinse with dH2O, aspirate or drain, gently blot remaining water with a strip of filter paper (avoid contact with the gel surface), pour stacking gel.
Thank you so much, Bruce!! :) I have another question if Bruce or someone else could hopefully answer: would 30% acrylamide solution or TEMED go bad over time? I bought these reagents a while back and stored in the fridge and thought it would be OK, but gel has not been working properly in resolving proteins... ^^:: (I made fresh ammonium sulfate and 10% SDS)...
TEMED is pretty stable. I don't keep AP solutions more than a week. I only make up about 1 ml at a time. Acrylamide hydrolyzes. The fresher the solution, the better. I make up the acrylamide solution (30:0.8 acryl:Bis) from powders and try to use up my stock within a month. The commercial suppliers indicate their solutions are stable for longer, but I have never used a commercial acrylamide solution.
Thank you again, Bruce. Maybe 30% acrylamide solution may be the problem. ^^;; I did not want to buy the powder, knowing that it is a health hazard... but may be that is something I can consider buying. I just bought a bunch of liquid bottles, and well, a little embarrassed to tell you, but it was from 2012. ^^;; Thank you so much for sharing your insight!
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