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Pull-Down Assay, EMSA Assay: recombinant protein vs nuclear extracts

EMSA Recombinant Protein Pull-Down Assay Protein DNA Complex Proteomics

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#1 AntonioSillo

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Posted 07 April 2015 - 03:55 AM

Hi to all,

It's The first time in this fantastic forum!

I think that you can help me.

I'd like to study by NMR a protein-DNA complex.

It's well known, as reported in several papers, that a protein found in mouse embryonic fibroblast interact with a portion of 30 bp of DNA.

So I've repeated with EMSA Assay this experiment using Human Embryonic Nuclear Protein extracts.

I had positive results. I repeated this experiment with the recombinant protein known to interact with the 30 bp DNA probe, but I had no positive results. I tried to study this interaction also by NMR, but still no trace of any interaction.

I tried to digest the pulled down protein within the nuclear extracts, in order to look for any post translational modifications..., but the nuclear protein seems not to undergo to any PTM.

I tried another assay: I used Streptavidin coated magnetic beads, and I observe a positive result both with recombinant protein and with nuclear protein.

Why??

Furthermore the protein, eluted with various methods (SDS Boiling, Tris glycine.....) Seems to band at a MW compatible with a dimer?

How is it possible since I've used denaturing conditions?

Thank you



#2 mdfenko

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Posted 07 April 2015 - 04:27 AM


Furthermore the protein, eluted with various methods (SDS Boiling, Tris glycine.....) Seems to band at a MW compatible with a dimer?

How is it possible since I've used denaturing conditions?

 

do you also use reducing agent? if not then disulfide bonds are probably responsible for producing the dimer.

 

if you have then you may be boiling too long. even in the presence of sds, overboiling can cause aggregation. you can heat at 60-70C for 10-20 minutes instead of boiling.


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#3 AntonioSillo

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Posted 07 April 2015 - 04:34 AM

thank you for replying me, yes, I've used DTT + SDS or  Glycine-HCl pH 2 in my elution buffer.



#4 mdfenko

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Posted 07 April 2015 - 05:01 AM

when you use pH 2 glycine, do you neutralize immediately after collection? the protein may not be stable at low pH.


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#5 AntonioSillo

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Posted 07 April 2015 - 05:02 AM

yes, i neutralize with TRIS



#6 mdfenko

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Posted 07 April 2015 - 05:07 AM

do you then dialyze or otherwise reduce the ionic strength of the solution?

 

high ionic strength can cause all sorts of complications with page.


talent does what it can
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Also tagged with one or more of these keywords: EMSA, Recombinant Protein, Pull-Down Assay, Protein DNA Complex, Proteomics

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