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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.


Why I got two RNA bands??

Why I got two RNA bands?? - Electrophoretic Mobility Shift Assay Forum

Why I got two RNA bands?? - 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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Old 01-06-2009, 05:45 AM
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Default Why I got two RNA bands??



Hello, I'm glad I found this forum!

I tested the RNA after in vitro transcription and 32P labeling and I got one band. But every time I did EMSA, I always got an extra band above the RNA. It's not b/c of the shift since the RNA alone also has the second band. It could be the secondary structure? How to get rid of the second RNA band? I got the shift but the second band really annoys me...Did anyone here got the same problem and how did you solve it? Thanks.
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Old 01-06-2009, 08:22 AM
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Default Re: Why I got two RNA bands??

Hello there,
welcome to the forums

are you doing denaturing or non-denaturing emsa gel electrophoresis?

Could you post an image (just go to Go Advanced - Manage Attachments) so I can help you more.

cheers
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Old 01-06-2009, 09:35 PM
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Default Re: Why I got two RNA bands??

Thanks. Here is the image. The first and last lanes are negative controls, and the second band is very clear. I used 1% non-denaturing agarose gel. The buffer I used is 20 mM Hepes 8.0, 50mM KCl, 5% glycerol, 1mM DTT. 30 min on ice. I have another question, what is the purpose of using KCl, is it necessary? It seems MgCl2 is good for another protein I'm working on and it also made the shift of this protein further, but the second band is still there.

Another image is the probe I made (32P labeling). Without any RBB, there is no second band. So could it be sth wrong in the RBB?

One more question about the non-radioactive kit. I checked their protocol and it seems it's mainly for DNA-protein binding. Is it applicable to RNA? How to make some modifications for RNA binding?

Sorry I got so many questions here. I'm new to EMSA and I think I will be doing a lot.
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File Type: jpg Cmpp16 in vitro transcription copy.jpg (4.8 KB, 19 views)
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