Non radioactive EMSA!!!

[DNAIA]

Hi all,


I am doing non radioactive EMSA using acrylamide gel, but I am wondering how would I transfer the complex to my membrane and what kind of buffer do I need to use for transfer?

[mmorgan]

Do electro-transfer onto a DNA binding filter in TBE buffer. Or use 32-P labelling and save yourself some time.

[DNAIA]

Originally Posted by mmorgan

Do electro-transfer onto a DNA binding filter in TBE buffer. Or use 32-P labelling and save yourself some time.

Thank you for your reply. But my supervisor wants Biotinylation, and I did it but the transfer was the issue, also I am using RNA, do you think TBE is the suitable buffer and what about semi dry transfer do you think it will work better? also do I need to use the same buffer that I use for prapering the acrylamide gel?

[admin]

Hi I have done non-radioactive EMSA with transfer and you need a special membrane to bind the RNA during transfer, which one are you using as buffers aside that is the most important part?

TBE was compared to TAE in some publications I read, and the TBE was better but both need cold conditions during running/transfer

[DNAIA]

I am using PVDF membrane, crosslinking for 4 minutes, also I am using vectorlab biotinylation kit, I succeeded to do the biotinylation in my transcribed RNA with this kit.

I don't use cold conditions in running but I do in transfer, also could you please recommend the voltage/Am, my RNA is very big 9.6 kb.

Many thanks

[mmorgan]

Is PVDF the best choice for this assay? It will certainly retain protein but perhaps using a membrane optimized for nucleic acid might help (Hybond N works well for binding RNA for Northern blots).

[DNAIA]

when I did the titration of the biotinylated RNA the PVDF worked fine!

[DNAIA]

any suggestions!!