Emsa Trouble???

[chromosome11mutant]

Hi,
I'm getting no shifts in my EMSA, all I get is the P32 labelled probes at the bottom.
At first I thought the problem was with the Nuclear extract that I used against my probes. Howerver that wasnt the case.
I ran a parallel control (using my collegues lab. probe different from mine), together with my probes. i observed a prdicted shifts with here probe, but again no shifts with my probes.

It seems like a problem with my probesp; I cant understand what??
I double checked my probe designing, and then TESS and there seems to be many proteins that should be binding to my probes.

What do youy think might be the problem?? and how do I go from here???


Thanks

[kmunson779]

Are you sure your probes are being labelled? (that you're not just seeing unincorporated P32?)

How are you making/labeling your probes and purifying them?

Do you have a purified protein that you can use as a positive control?

[chromosome11mutant]

Seeing unincorporated P32 is highly unlikely, as these bands are sharp bands and are aligned with the parallel control lab. probe (same length).

No I dont have a purified protein, I'm trying to characterize the binding protein!

[admin]

Hello there,
welcome to the forums.

I won't say I am an EMSA expert, although I have been doing it for about 7 years.
There could be several problems:

1) your probe buffer may be inhibiting the reaction (this can happen in several ways including a) unincorporated nucleotides inhibiting binding b) salt concentration c) contaminants

2) your probe may be labeled differently than your partners. It could be labeled in the binding area or the label could be sterically inhibiting the binding of your protein.

3) Is your probe identical in size and sequence to your partners? This obviously would affect shifts (number of proteins (complexes) bound in non-denaturing shifts and size of protein(s) in denaturing shifts)

These are a few possibililites but we would need a bit more information on how the binding and probe creation were done.

please report back here to tell us more or how this unfolds.

Cheers