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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.


EMSA with tumor samples - Loading control

EMSA with tumor samples - Loading control - Electrophoretic Mobility Shift Assay Forum

EMSA with tumor samples - Loading control - 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 09-04-2008, 08:29 AM
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Default EMSA with tumor samples - Loading control



Hello everybody,

I hope somebody will be able to help me with this...
The thing is that I'm doing EMSAs with Breast Cancer tumor extracts and I'm obtaining quite nice results with NF-kB, but not such results with Oct-1 (as a loading control).
I'd like to ask if there are other transcription factors that can be used as loading controls and, if someone has experience with tumor samples, is there any TF that is "better" for Tumor samples than others?

Thank you very much for your time. Any comment will be wellcome.
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Old 09-04-2008, 10:26 PM
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Default Re: EMSA with tumor samples - Loading control

Hi Amazonic,
i am not sure i understand as usually "loading controls" are done in EMSA using the unbound probe.

One can easily quantitate either quantitatively or qualitatively the amount of bound probe vs unbound probe.

For example:

= -

- =

3 3

Both equal 3 despite the fact that there is more probe bound in lane 1. I believe that loading controls would be unnecessary with EMSA especially radiolabeled. Are you using non-radioactive detection methods for EMSA? Even with that you can use detection with a densitometer which still allows you to count luminescence qualitatively over time.

please let me know this is an interesting question
cheers
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Old 09-05-2008, 08:10 AM
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Default Re: EMSA with tumor samples - Loading control

Hi admin,

Before going into the issue I'd like to thank you for the quick answer and your help.

I use radiolabeled probes to perform EMSA, so I can do what you've explained. In fact, I already do, but in a qualitative way.
The "problem" is that my boss wants to have another transcription factor as a control, a TF that is supposed to be expressed the same way in all conditions. We were using Oct-1, and in cell line extracts we have good results, but not with tumors. So that's why I wanted to know if there's some TF to look at that is better than others in tumor samples.

I know that to use Oct-1 as a "loading control" it's not correct, in the sense that the reaction with Oct-1 probe is independent of the one with NF-kB (that is the one I'm interested in), because maybe I can make some errors when I pipette (I don't know if this is correct in english) the volume of extract or something.

Well, thanks again, and if someone has any idea of other TF that could work with Tumors I'll be glad if you let me know.

Cheers.
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