My protein of interest is of 29 kd. on ponceue staining low molecular weight protein doesn't get transferred and thus no band was observed in the blot after secondary antibody addition. I was using 10% gel and 20% methanol on towbins buffer. I added around 40ug protein per lane.
Hello Jyothi
1.Increase your acrylamide concentration from 10% to 15%.
2.I hope you are doing a semi - dry transfer.
3.Use a 0.2uM pore size PVDF membrane instead of the 0.45uM pore size.
4.Add 0.1%SDS in transfer buffer as it improves transfer efficiency for PVDF membrane.
Is the ladder getting transferred?
The image is not clear to me.
thank you for your response,
I was doing the wet transfer method and a nitrocellulose membrane for transfer.
the ladder is somewhat transferred but not very distinct.
Hello Jyothi
So try 0.2uM pore size PVDF membrane and use semi-dry transfer.
All the best!
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