We have recently switched from chemi to florescence for the imaging of our Westerns. We have also recently had our Trans Turbo Blot serviced and fixed. We have been noticing many speckles on our blots and we don't believe it is any errors in the blotting process, but rather something in the transfer. Our guess it is polyacrylamide residue melting to the nitrocellulose membrane. Has anyone had any experience with things like this happening?
The conditions used in Trans Turbo Blot procedure are prone to produce much more heating of the gel and the membrane than the regular liquid (wet) transfer protocol (our experience). We could not get a good transfer of our protein using Trans Turbo Blot (but it may depend on the each particular protein). I can't tell you that it's caused by polyacrylamide residue melting to the nitrocellulose membrane or some strong denaturation of the proteins that might make them incapable to leave the gel, but the heating factor is definitely involved. I would recommend to switch to usual liquid (wet) transfer system.
Increased heating of the transfer buffer might be due to abnormally high salt concentrations. Does your lab prepare its own transfer buffer or was it purchased? If the first case is true I would suggest to prepare fresh transfer buffer and re-try the blot.
Good luck!
Cheers, Christian
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