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EMSA problem


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#1 marylka

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Posted 19 August 2004 - 09:17 AM

Hi, i have a problem with binding during EMSA!
i checked nuclear extract by WB and i know that it contains my transcription factor
probes are labbeled beautifully
but i cannot see any bands just free probe, seguence is correct !
i checked so many times
please help me :o

#2 kant0008

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Posted 19 August 2004 - 03:37 PM

Hi,
could you specify your conditions? Do you use a kit? There are a few things you could try, like changing protein amount, running at 4C and no loading buffer, making sure your probe is ds, etc. But we need to know what methods you are currently using

#3 marylka

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Posted 20 August 2004 - 07:52 AM

Hi,
could you specify your conditions? Do you use a kit? There are a few things you could try, like changing protein amount, running at 4C and no loading buffer, making sure your probe is ds, etc. But we need to know what methods you are currently using

Thanks a lot! these are my details:
I'm doing electrophoresis in RT with 1xTBE, 0.5xTBE or even 0.25xTBE - no difference, gel is regular 6% polyacrylamide
binding buffer is 10 mM Tris 7.5, 50 mM NaCl, 1mM DTT, 1mM EDTA, 5% glycerol
proteins concentration - i tried from 5 ug to 50 ug - just more smearing
i checked 5 ug on WB- there was a lot of my protein
i wil run reaction without loading buffer, thank you
what's your other suggestions?
;)

#4 kant0008

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Posted 22 August 2004 - 10:11 PM

Hi,
try running your gel at 4C if you have the option to do it, your protein-DNA complex might be labile at RT.
Also if you are getting a smear (nonspecific binding?) try decreasing your protein amount, try 1ug or even less. Also with a smear sometimes it helps to add extra non-specific competitor, like polydIdC-polydIdC, try 0.2ug for every 5ug protein. If you get no bands at all, then don't add competitor at all, see what happens.
Good luck!




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