Protein stuck in the origin of resolving gel - (May/26/2015 )
My protein size is 260kDa and I am using 5% stacking gel and 7.5% resolving gel. After wet tank transfer, I am seeing that my bands positive for my protein are way up at the origin of the resolving gel. Please provide suggestions to improve the run. thanks.
Lower your gel percentage. I would use a 6% gel for this.
Also check your running buffers, throw out old solutions and prepare fresh.
Yeah use a 4% stacking gel.
It is possible your protein is aggregating in SDS. This is not unusual for membrane proteins if that is what you are working with. Are you boiling your samples prior to running them on a gel? If so, try not boiling. If you are not, try boiling. Also try with and without reducing agent.
Also consider sonicating your sample briefly prior to solubilization in SDS. This could be particularly helpful if you are running whole cells. Sometimes intact DNA can cause samples to be a little viscous causing them to run awkwardly on a gel.
also, instead of boiling you can heat at 60-70C for 10-20 minutes, sometimes proteins will aggregate when boiled in the presence of sds for too long.
Thanks for all your suggestions. I repeated with 4% stacking and 6% resolving gel, tried boiling and also at 37C, still I see my protein at the origin of resolving gel, worse yet, the 250kDa protein std marker is also at the top. Please help.
If your standard 250kDa marker is stuck in the stacking gel, something is wrong with your gel, your running buffers, or your gel box.
What voltage are your running? What amperage?
What is the pH of your stacking and resolving gels?
Try borrowing someone else's gel box to see if that is the problem.
Or procure a commerically casted gel to see if your home-made gels are being made incorrectly.
Or double check that your running buffer has the right concentration of glycine, tris and SDS. Checking the pH is a quick way to do that. If it's not 8.3, then re-make it.
how are you preparing the running buffer?
do you adjust the pH? if so then don't, this will introduce salts into the buffer and they will interfere with migration. laemmli running buffer should not be adjusted. just use the components as described in the protocol.
No I usually don't adjust the pH of the running buffer. I just re-checked the pH of the 1X running buffer it is 8.53.
try preparing fresh running buffer. make sure you use the same form of tris, glycine and sds (in fact, you may want to use a newer lot of sds, the old one may have decomposed).
if you want to check the pH then do it before you add the sds.
I prepared fresh running buffers, Tris buffers for stacking and resolving gel and still I see a faint 250kDa marker in the interface of stacking and resolving gel. I am waiting for commercial gels now. Please see this pic.