I will try to be as thorough as possible. So it's been over a year or so and I went back to pouring various % SDS Page Gels and I keep running into problems I didn't have before as seen below. The gels are running somewhat fine in the beginning but then when they are about half way through the proteins begin to "smear" for seemingly no reason. After this gel I replaced the TEMED (it was quite old) but I'm still running into problems. I tested the samples against a pre-cast and they looked fine but my gels still are polymerizing "weird". I changed to new TEMED, made fresh 10% APS, fresh 10% SDS page filter sterilized buffer, the 40% Acrylamide is old but that stuff never goes bad apparently, the only thing I wasn't sure about were the Tris-base buffers. So I made my mixture per protocol and let the gel sit for 30min with a dH2O layer on top so it wouldn't dry out then added the stacker, added the comb and let it sit for 20min before wrapping in a damp (dH2O) paper towel and wrapping in plastic wrap and putting at 4C over the weekend. I ran two gels w/identical samples monday and the gel in the back ran slightly faster than the gel in the front then they started to run slightly uneven and half way down they just started to smear. Even the ladder stopped half way through but the die front went all the way to the end of the gel (120V 90m). I checked and the 1x running buffer still covered the wells so I am at a loss as to what is going on any help would be greatly appreciated!
Hello Nathan,
Since your sample is running properly in the pre-cast gel, you need to make new buffer stock (1.5 M Tris, pH 8.8 for separating gel and 1 M Tris, pH 6.8 for separating gel) and also acrylamide mix. During storage, acrylamide and bisacrylamide will slowly convert to acrylic acid and bisacrylic acid. It is better not to use an acrylamide mix that is more than two to three weeks. TEMED it pretty stable.
Overlay the gel with saturated iso-butanol instead of water. take around 50 ml of iso-butanol in a bottle and 1-2 ml of MilliQ-grade water. Shake. Let settle. If you don't 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 100-400 ul (depending on gel size and thickness) of this as your overlay solution. When ready to add stack, pour off the overlay. Rinse the gel surface with MilliQ-grade water, place gel on its side on the bench, use a small strip of filter paper to remove remaining water (don't touch the gel with it), place upright, add stack solution and comb.
Best
pramod
Thank you for the prompt response. I remade the pH 8.8 and 6.8 buffers yesterday as I'm sure they were at least a year old or more. The 40% acrylamide mix we have is premixed and kept at 4C per instruction although I know the bottle in use has been going for over 2 years. I will look into the isobutanol, I had read that elsewhere but I have not had the chance yet to try it. Thanks for the help!
Hey there!
I experienced similar patterns while pouring gradient gels. While I was pouring the buffers between the glasses I moved the tip of my pipette from left to right, left to right, until I filled everything. I guess that was my problem. I changed the way I pour the gel between the glasses. Now I fill it in at one edge, I do not move it.
I suppose since the gel is not evenly running down the glass it somehow polymerizes a little bit at the back or there are somehow remainings that stick to this pouring pattern. The remaining gel is covering the pouring pattern and maybe polymerize a little slower or just overlays the pattern.
Do you use a lot of TEMED and APS? (I use 20ul 10% APS and 7ul TEMED) If yes, your gel will polymerize faster.
Consider to reduce the volume of those two and pour the gel faster between those glasses (at only one edge).
Since I pour my gels like that I dont have this pattern problems anymore.
Best
Lisa
- Badges
- Science method