Hi, I ran two gels couple of days ago and did not get good bands. I initially thought that it was my secondary that's not working well. But when I checked the gels I realized that there were thick bands at the low molecular weight marker levels.
Every time I do transfer I would check if the ladder is transferred onto the blot (that's how I determine if the transfer is successful). The ladder was on the blot but why are the proteins not transferring??
Normally the smallest proteins are transferred quickly, so this problem seems odd to me. Regardless, I always aim to have my protein of interest in the middle of the gel, adjusting acrylamide concentration. For a small protein I would increase the gel concentration to 15 or even 18%. I am working with a 25 Kda protein and I use 15% gel in order to get my protein in the middle of the gel. Good luck!
Hi Elena,
As Fernanda lead off with, small proteins usually transfer fastest. It is possible you are allowing the transfer to proceed for too long and your proteins are going out the back side of your membrane.
You can try decreasing time of transfer or sticking additional membranes behind your intended membrane to see if they are going too far.
Good luck!
My protein of interest is beta actin and im using 10% gel. I would usually do 1 hour transfer for proteins less than 60 kDA and it seemed to work fine for me. I dont know why it didnt work this time. How is it possible that the ladder was transferred but the proteins werent??
I hope you are not running the gel at most end, so it depend upon the protein size you are looking for. I agree with the above reply, you shold decrese the transfer time, better to use some coloured protein marker for reference .
I hope this will help you
If the proteins went out of the blot, they should end up in the buffer right? Not back in the gel. My problem here is that low molecular weight proteins are not getting transferred, and how does reducing the transfer time help with that. It doesnt make any sense.
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