I am a beginner of western blot and from some days I have been facing a lot of problems . My experimental protein size is 124kDA and I choose Beta Actin as a loading control which is small molecular weight protein.Generally, it is suggested that during high MW transfer, one should use wet transfer.When I have transferred the entire gel into PVDF membrane (0.45um) at 60V for 40mins, there was nothing( ladder) left neither into the gel nor gel and pads. First I thought that the time is too long for the smaller one, so I cut the gel into two pieces and tried to transfer the bigger one at 60V for 40mins and smaller one for 30mins but I got the same result.There was no sign of ladder into the membrane. My transfer buffer composition is 20% methanol, 48mM tris-base, 39mM glycine and SDS 0.1% final concentration.Is there any problem in buffer composition or the transfer parameters?
Hello Maynak,
did you do an immunodetection with one of your membranes? What brand and model do you use for transfer? Check the power supply with a standard SDS-PAGE.
Your buffer and transfer conditions seem plausible to me.
Cheers, Christian
@ Christian
I did the immunodetection but it did show nothing. We use Bio-rad wet transfer apparatus and Merck PVDF membrane.Thank you
Make sure you have the electrodes set up in the correct polarity. If the polarity is reversed, the proteins will transfer in the opposite direction from the filter and end up in the transfer buffer.
@ Adam,
We have checked the electrodes position every time .
@ Ning,
We generally use Biocore life sciences ladder.We have developed the blot but there was nothing.Is there any problem in buffer composition?
Thank you
We generally use 20% methanol but I have tried with 15% also.I do not able to detect my protein and ladder also did not transfer.Sometimes half transfer, sometimes there was nothing. If you run a gel 90v for 100min and detect a high molecular weight protein , can you able to detect a low molecular weight one in the membrane? Because at that time it has already passed through the membrane. I use 0.1% SDS also.
The problem might be related to not pre-wetting the PVDF membrane in methanol before soaking in transfer buffer. Always check transfer with ponceau. No ponceau stained bands means no transfer to the membrane. Either the membrane was not pre-wetted, or the proteins went the other way (reverse polarity).
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