So when I prepare my 10% SDS gel for Western blot, I see that my resolving solution is perfect but my stacking solution does not seem to be working properly. So when I take the comb out, the wells are not formed and that makes it impossible to load any samples.
I leave my gells in the fridge overnight with the comb in and gently take the comb out before loading.
MY stacking solution polymerizes in the tube and anywhere else in the gel but around the combs it seems. The Temed we use is quite old. However, as I use Temed and 10% ASP in both resolving and stacking buffer, it made to think that I have to rule out if one of these are an issue.
The cmb seems to have the right thickness too.
I appreciate your thoughts and experience on this.
Thank you,
Vahid
Hi,
First of all, you should check whether the pH of stacking gel buffer (1M Tris-HCl, pH 6.8) is appropriate or not. Second, 10% APS should be freshly prepared. Third, Temed should not be too old. But the last two reasons ruled out because these components also used in the preparation of resolving gel. So, please you need to focus on the first reason.
I think the question might be the APs or temed, since the stacking gel is 4%. you could try add more APS and temed (doubled) into stack gel to see what happened.
as TEMED gets older its polymerization efficiency becomes weaker. You can add two to three times of TEMED. It will polymerize.
Second thing while casting the gel, reagents should not be cold, bring it to 37 degree prior to use.
Thank you all for your replies.
I actually doubled the Temed and APS and still had the same problem. I ordered a new Temed now.
Jayant Dewangan Do you mean to bring Temed and APS to 37 degrees before adding or that also applies to resolving, stacking buffer, water and DTT too?
@Vahid
I ment only resolving and stacking buffer, not the TEMED and APS.
Even after adding extra volume of these reagents still the problem persisting means these reagents might expired. Using the new one may solve your problems.
If possible I recommend to prepare Tris buffer also fresh and maintain required pH.
Try to work at room temperature and check that your stacking gel don´t has leaks during polymerization and .. do you put something between both gels? like alcohol or water?
Rocio Gaytan Thank you for your reply. I do work at room temperature and did not see any leaks so far. I don't think I put anything between two gels? Is that something that might help?
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