I am working with a family of low molecular weight proteins (around 22 kDa). I have optimised the conditions for a good separation in SDS-PAGE electrophoresis by Bis-Tris Gel and MES running buffer. However, in our lab we have the iBlot 2 transfer equipment performing transferring under dry conditions and I believe that I do not succeed the right transfer. I am using 20 mV for 7 min and when I stained the gel with Coomasie staining I could see that there were proteins left in the gel both low as well as high molecular weight proteins. I did the transfer for 7 min in 25 mV but then I think that I lost my proteins as the antibody could not detect the protein fragment and the when I stained the gel after transfer the low molecular weight proteins were not there. Does anyone have any recommendation regarding the proper conditions of dry transfer for low molecular weight proteins? Does anyone could help me understanding the parameters of time and voltage application in dry conditions for low molecular weight proteins?
Thank you in advance for your help
Hi,
Did you check with increased time, for example 20 mV for 15 or 20 min?
I use Transblot turbo for 15 to 20 minutes for several protein around ~20kDa.
Good luck.
Hasan Al Faruque
Hi Hasan, with the iBlot 2 transfer system when I used 20mV and I exceed the 7 min, then I saw a blue colour (this is an effect of time and voltage application on copper stacks that iBlot 2 transfer has in its system). So, I did not increase the time. Probably, it would be a good idea to try with less voltage and longer time periods. May I ask you the voltage that you applied for Transblot Turbo for your small molecular weight proteins?
Hellow Danai,
For Transblot Turbo, I used 25V 1.0 Amp for 20 minutes and it works well. However, iBlot 2 has limit for voltage and time adjustment, therefore, you should increase time after adjusting volt as recommended by manufacturer.
For example iBlot2 has P5 10 V 7 minutes (default) 25 minutes (run time limit).
Hello Hasan,
Do you use proprietary packs from Bio-rad for Transblot turbo or do you use any homemade buffer? I tried Bjerrum-Schafer buffer(48mM Tris, 39mM glycine, 20% methanol, 0.1%SDS. I also reused the pads provided by Biorad by rinsing them in distilled water and soaking in the transfer buffer 10min before transfer. I followed the High MV, 10min settings. The Amperes dropped from 1.3A to 0.4 in cassette A and to 0.8A in cassette B (one gel in each). When doing 2 mini gels at a time in one cassette, it dropped to 1.9A from 2.5A.
Did this happen to you ever and what precautions did you take.
Kindly advise.
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