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Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


Non radioactive EMSA!!!

Non radioactive EMSA!!! - Electrophoretic Mobility Shift Assay Forum

Non radioactive EMSA!!! - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


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  #1  
Old 01-24-2012, 08:12 PM
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Default Non radioactive EMSA!!!



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?
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Old 01-24-2012, 08:21 PM
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Default Re: Non radioactive EMSA!!!

Do electro-transfer onto a DNA binding filter in TBE buffer. Or use 32-P labelling and save yourself some time.
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Old 01-24-2012, 10:07 PM
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Default Re: Non radioactive EMSA!!!

Quote:
Originally Posted by mmorgan View Post
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?
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Old 01-25-2012, 12:23 AM
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Default Re: Non radioactive EMSA!!!

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

Last edited by admin; 01-25-2012 at 12:24 AM.
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Old 01-25-2012, 11:57 AM
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Default Re: Non radioactive EMSA!!!

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
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Old 01-25-2012, 06:25 PM
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Default Re: Non radioactive EMSA!!!

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).
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Old 01-26-2012, 06:33 AM
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Default Re: Non radioactive EMSA!!!

when I did the titration of the biotinylated RNA the PVDF worked fine!
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Old 01-29-2012, 01:13 PM
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Default Re: Non radioactive EMSA!!!

any suggestions!!
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