Hello all,
I am used to using the TurboBlot for Western Blot and had no issues with exception of transferring high MW proteins. That's why I wanted to try the Wet transfer method. However, everytime I try it, the lower bands appear so badly on the membrane (the images are total protein blots). What could be the cause?
I am running the system (Biorad Criterion Blotter) with a constant voltage of 100V in a cold room (the problem is not overheating). The buffer I use is the recommended by Biorad (I do it myself). While I am running, I put an ice pack inside together with a magnetic stir. Could it be the magnetic stir motion causing this?
Thank you.
Dear Filipe Pinheiro , I assume this only happens during transfer and the gel itsself would look normal after running? In that case only thing i can think of is that there might be a problem with the contact between membrane and Gel. If not completely tight, the bands may diffuse away? You could check: are the pads/Whatmann old or thin/patchy, and maybe need replacing? are there any air bubbles between gel and membrane? is the membrane properly wetted everywhere and didn't dry out? Is the tank properly filled to cover the whole sandwich? I don't think the stirrer is the problem if everything is well assembled.
Dear Filipe Pinheiro this happens for my sample also, go for overnight transfer for 16 hours at 20 volts, which have solved the problem for me, and also check the pH of the all the buffer which you use (stacking, resolving and running buffer)
Thanks
Venketsubbu Ramasubbu The buffers that you mentioned are for the SDS-page. I am not having problems at that stage. The problems are at the transfer of the gel to the membrane.
Daniela Liebsch Thank you for your answer. Yes, the gel is totally fine. I always activate the gel for total protein and everything seems normal. I will try to troubleshoot by the possibilities you mentioned
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