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Electrophoretic Mobility Shift Assay


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3 replies to this topic

#1 Aravin

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Posted 13 February 2011 - 07:06 PM

Hello,
I performed an EMSA using a kit. I had 3 wells, one was the positive control provided by the kit, and the other two were my nuclear protein samples incubated with a biotin-labelled probe. After I developed the film, I was only able to see the free probe and shift of the positive control. However, there was no signal from the 2 samples and most surprisingly, there was no free probes observed in those wells. Do you think it is a problem with the probe and maybe degradation occurred? I'm confused because the probe from the positive control worked but not my probe. Please let me know what you think.

Thank you!

#2 pcrman

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Posted 15 February 2011 - 08:52 PM

It looks like your probe is probematic. How did you prepare your probe?

#3 Aravin

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Posted 21 February 2011 - 08:00 AM

It looks like your probe is probematic. How did you prepare your probe?


We prepared the probe using a kit. I tested the probe by itself by blotting it on the membrane and developing it. I actually got a signal. So the probe is working fine, but this problem is now more confusing.

#4 Aravin

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Posted 06 March 2011 - 10:38 PM


It looks like your probe is probematic. How did you prepare your probe?


We prepared the probe using a kit. I tested the probe by itself by blotting it on the membrane and developing it. I actually got a signal. So the probe is working fine, but this problem is now more confusing.



So in my next experiment, I just ran the probe in the EMSA and the probe DID work. It seems to be something wrong with my nuclear extracts or my emsa protocol. Any ideas?




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