Hi guys,
So while carrying out PAGE, following the addition of propanol alcohol, the surface becomes smooth, but not straight, it usually starts to take a lower angle near the end. It's not a huge deviation from the straight line, but it is noticeable when working. Is it an issue I should be concerned about? Could it affect the electrophoresis? It happened more than one time by the way, and hence my question. Many thanks for your time.
Hi Mostafa this has happened with me as well and it tappers at the end. the gel is more cohesive than glass so its fine as long as it only at the ends (not closer to your well)
Also, how long is your gel solidifying time? Try to play around with TEMED if you are getting a steep bend.
I believe you should not have any issue with the gel run. Hopefully, you wont be loading the extreme left or right lane of the gel. I did find the similar thing. As suggested by Abbas you can play with TEMED to have a longer polymerization time.
While loading the wells try to fill the empty or left out wells with SDS sample buffer because I myself have found difference and my protein lanes were either somewhat tilted or distorted. with all wells containing the loading buffer, you can see the dye front moving in a uniform straight line and gel looks very beautiful. :)
Hi, I also hate the angle because can affect the running. I will follow all the advices just given, for me the problems are probably the glasses and the sponge as Sarah said. I would try to clean really well the glasses (if you cannot change the glass less used) with alcohol. For the sponge I cut a piece of parafilm and put on the sponge, in general if I have old glasses with microchips.
I do not know if you have problem with your solidifying time, I find that it is better to change frequently APS (together with TEMED concentration if you want), if you follow the Sanger recipe, with fresh APS, it is always take no more that 30 minute.
TEMED would solve your problem, I once had this problem and I used TEMED and all went well.
Sounds to me like your gel is solidifying pretty fast. If that's the case play with the amounts of TEMED and APS used. Also consider the acrylamide:bisacrylamide ratio in doing so.
(1)100% 2-propanol is not suitable to faltten the surface of separating gel, but 50%(v/v, approx.) 2-propanol is better, I think. Pour 50% 2-propanol gently on the surface of freshly poured separating gel at low angle with laying gel-glasses, then stand gel-glasses again.
(2) The concentration of TEMED or APS might be too high. i.e. Too fast polymerization.
To follow others, I would suggest altering your APS concentration in the gel solution. Lowering the concentration will increase the amount of time required for the gel to solidify. However, this is only a suggestion if your gel isn't leaking out of the one side-fix the leak first. It is important for all of your samples to enter the resolving gel at the same time.
I use 100% 2-propanol, ~ 100 microliters, to flatten the gel surface, and pour it off before I pour the stacking gel.
Dear Moe,
We use 100 % isopropanol its always straight. We use the attached concentration of chemicals may be useful.
All the best.
Moe
Find a leveling stand. It could be that your bench is not 180o .
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