Degradation of probe in EMSA - (Mar/07/2008 )

Hello everyone,

I am trying to do "Band shift Assay" (EMSA) for NF-kB translocation but at certain conditions I see that my signal is much weaker than controls, that also occurs with the free probe that I usually see at the end of the gel. It is also weaker than my probe. I am thinking that in someway the radioactive oligo is being degraded or dephosphorylated. My buffers contain EDTA so I do not think that would a problem. As for dephosphorylation, I treated the samples with NaVO4 but still I got the weak results. What else could it be?

I appreciate your help
Zaanaa

I'm not exactly sure what your problem is from your description. It is highly unlikely that your probe is being degraded or dephosphorylated. I have done NF-kB EMSAs in many cell types and have not encountered these problems.
If you are losing probe then it may be caught in the top of the gel. Do you only lose probe when extract is added? Probes can degrade over time, is it old? Did you run your gel too long and run off the free probe? If it is a new probe maybe the oligos didn't anneal properly or label properly.
If possible, include a photo of the entire gel in a post.






Hello everyone,

I am trying to do "Band shift Assay" (EMSA) for NF-kB translocation but at certain conditions I see that my signal is much weaker than controls, that also occurs with the free probe that I usually see at the end of the gel. It is also weaker than my probe. I am thinking that in someway the radioactive oligo is being degraded or dephosphorylated. My buffers contain EDTA so I do not think that would a problem. As for dephosphorylation, I treated the samples with NaVO4 but still I got the weak results. What else could it be?

I appreciate your help
Zaanaa
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