Hi All, I have been doing a wet transfer for my 10 % gel in a NC membrane for a protein at 42kDa at 100V for 1h with a freshly prepared transfer buffer substituted with 20% methanol. After my transfer, I stained my membrane with Ponceau. I could not see the abundance of proteins , rather it was a very faint band of just 42 kDa. When I proceeded with the probing of the membrane, the loading control ( Tubulin ) band didn't appear at all, although the Tubulin was a freshly prepared antibody, being used for the first time. Is it like, if we re use the transfer buffer , we don't get a good transfer? Or is it good with a low methanol concentration for low molecular weight proteins?
We allways use 20% methanol for wet transfer, and it works fine. But we pay attention to the current, not voltage. 2h of 400 mA for ~40-60 kDa proteins usually is optimal. Prolong the time proportionally if your power supply cannot provide 400 mA - overnight at 80 mA is also acceptable.
Voltage is the driving force for transfer. Current is the limiting condition. The larger the format for the gel the more current, which scales by area. More current means more heat, and a lower voltage. There are also secondary effects due to buffer strength. In general you want to keep your transfer buffers at low concentration to help facilitate protein transfer, but not so low that the protein kicks out of solution. You can compensate for lower voltage by increasing the time. Keep V-h constant. Are you using a manufacturer's kit or home made? If a kit double check the instructions and make sure that you are following religiously. If home made, then pull a set of kit instructions from the web and double check your recipes and procedures.
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