EMSA problems - (Apr/04/2006 )

Hi,

I need help in doing my EMSA, I have two main problems:

1) I can't use polydIdC or salmon sperm because band of interest disappeares while, without it, I can modulate it with specific and aspecific competitors.
someone told me it is because my nuclear extract is not really good. do you have any advice to make a better extraction?

2) my band is sometime well evident and others looks like a smear that anyway disappear with the specific competition

I am working on Interferon receptor factors and the pH of binding buffer I am using is 7.5 while my running is made at 8.5. Non denaturating Gel is composed 6% acrilamide/bis 60:1, and I run it at 150 V.

thanks, for any suggestion, I have no more ideas to play with.

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I could not answer the first question , i always use purified protein ...


Secon question. SmeARS TEND TO APPEAR IN TWO CASES:


1) overwharming of the gel. iGenerally is recomended to run the gel in a cold room (4ºC). If this is not possible try in a lower voltage ( 80-90 V). Gel runs slowly but you avoid overwharming.

2) the running buffer (TBE) is not really fresh. Salts tend to percipitate causing this kind of smears!
So make sure to renovate every two weeks or so.

Good luck!

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well, if you could post your nuclear extract protocol?

I think it sounds as though your extract isn't good

are you freeze/thawing it multiple times? are you using old extracts? is your protease inhibitor good? are you careful to normalize amount of total extract in every lane? inconsistent amounts or degraded extract I think would lead to smears in this case

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I tried two different extraction protocols but the problm was not solved:
nakamura protocol 2002 needs a low salt composed of 10 mM trisHCl, 1.5 mM MgCl2, 10mM KCl, 1mM DTT, NP-40 and a mix of antiproteases, in ice 5' , then pelleted at 12000rpm 20' and sospended in a high salt solution 30' at 4°c with a slow movement, then I aliquote the SN recovered after a centrifuge at 12.000 rpm 15' . I quantitate protein content with Biorad, don't thaw more then once the sample stored at -80°C, I add other antiprotease mix during the binding with my probe and run my gel in cold room for not more then to hours at 150 V.
my binding is made at room temperature for 45' , I tried in ice but it doesn't work.
the other extraction protocol I tried comes from Schreiber 1989, it uses a low salt composed of hepes, HCl, EDTAand DTT + antiproteases mix added just before use, incubate on ice 15', then added NP40 at the cellular sospension, vortex 10'' then on ice for extra 2' and centrifuged at 12000rpm for 30''.
wash the pellet with the same solution ,and add 3 x pellet recovered volume of high salt solution composed of hepes, NaCl, EDTA, DTT, and mix, on ice for 1 hour with strong and repeated vortex.
Supernatant obtained after 5' centrifuged has been aliquoted in precooled tubes at -80°C.

my running buffer is a tris glycine buffer ph 8.5

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