Hi everyone, I've tried to transfer initially using a 20% methanol transfer buffer using 250mA max current for 70 mins, all I got was transfer of mid-high range proteins, I suppose all proteins below 20 kDa escaped the membrane from the other side. Anyone has any experience with blotting 5kDa +- peptides and can share tips?
Hi Eli Farber,
I will share with you some tips that can help you with optimal analysis:
Remember that optimizing transfer conditions for small proteins can be a trial-and-error process, so don't give up.
Good luck
Hi Jakub Knurek
Thanks for the prompt response!
I have been using 20% acrylamide gels as you've recommended, tips 4 and 5 I've also applied before. The first time I ran the experiment it was 70 minutes of 250mA, and the following experiment was 30 minutes of 200mA. In the former I've got a transfer of only mid - high range kDa proteins, and in the latter it seemed the transfer was not efficient whatsoever. I suppose the conditions should be met somewhere inbetween these two.
I might try as you suggested using constant voltage, how do I know what is the equivalent amperage to 100V in a wet tank transfer apparatus (BioRad)? It would be useful to know it to compare to my previous trials.
Thank you very much again for your time and effort to help my research!
Eli Farber, it depends on the power supply you are using. If you are using any PowerPac™ from Bio-Rad, you should be able to set one constant value (amperage or voltage) during the transfer. You can select into this parameter in the options before. I remember that in the case of the equipment I used, the locked parameter was indicated by a glowing LED on the front panel of the unit.
If you still have problems with this setting, check the guidelines in the power supply documentation from the manufacturer at
www.bio-rad.com/webroot/web/pdf/lsr/literature/4006213.pdf
Best for You
Just to update, I've successfully transferred the small proteins / peptides from 20% acrylamide 1.5mm gel to a PVDF membrane using 250mA for 45 minutes. In case anyone googles this question :)
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