In western Blot,I have nothing transferred on the membrane& the gel at the end was dry.does the heating do tha

Question

I was doing Western Blot, it is my fourth time, last 3 times was successful but this one, the gel was dry, the membrane was like plastic and nothing transfered. the ice was a little bit hard, so I think it is a heat problem. Does the over heating do that ?

Thank you in advance for your time and your answer !!

Karim O. GAMAL.

Answer 1

It sounds like your gel dried out before you set the transfer.  What kind of a membrane are you using?  Are you doing a semi-dry or wet method?  If you are doing a wet method, you should not have anything dry at any point (membrane wet with MeOH then washed, gel incubated in transfer buffer before being put together in the apparatus).  To be on the safe side, instead doing a super-transfer (which is what I am guessing you are doing since you are using ice), do an overnight transfer at 24 volts, in a cold room.  And if your lab has equipment for a wet method, try doing that.  

Good luck and let us know how it goes!

Answer 2

check the power of the current you are running in western blot. Are you sure you set it to protocol standards? If you run the charge to high, you might end up melting your gel. The iceblock you put in can only regulate your buffer temperture for so long before the it gets too hot. It might be overheating caused by a charge higher than protocol standards.