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EMSA not detect a bindind


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#1 alchemic

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Posted 10 March 2015 - 01:45 AM

Hi,
 
I want to determined the Kd of the binding of ~400nt long RNA and a peptide which has 13 aminoacid residues.
I did a filter binding assay which shows a binding and Kd ~17uM.
 
 
As a assume that the peptide can bind in multiple sites, I want to see this in EMSA. I did 4% PAGE (acryl:bisacryl 80:1) native with 0.5mM DTT and 5% glicerol, 0.5x TBE  and run a gel in 4C but I don't see any shift using the same range of peptide concentration as in filter binding assay.
 
 

My peptide has a Iso-electric point: pH 8.5
The binding conditions are: 10mM Tris-Hcl, pH 7.4; 25mM KCl; 0.5mM DTT; 50ug/ml BSA;  RNase Inhibitor ; incubation 30min/ 4C. I assume that since I see the binding on the filter, the conditions of binding are OK, and the trouble is with electrophoresis.
Could it be that the peptide is so small, that I would not see a shift with a 400nt long probe? I can not shorten it.

Any advice on the electrophoretic conditions? Would agarose be better that acryalmide gel?
 
I would be grateful for any advice.
 
Alchemic



#2 mdfenko

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Posted 11 March 2015 - 04:27 AM

your peptide has a high pI of 8.5

 

the pH of the electrophoresis buffer should be higher to ensure migration from negative to positive electrodes or lower for migration from positive to negative.

 

a pH too close to the pI will inhibit migration.


Edited by mdfenko, 11 March 2015 - 04:28 AM.

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#3 alchemic

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Posted 11 March 2015 - 07:38 AM

Thanks for the answer.

 

 

On EMSA I don't see a decrease of free RNA, so it can be a problem with migration of protein due to pI. But I rather assume that RNA dissociate from peptide after binding, in wells? when pH of TBE, gel is somehow  wrong.

 

Would it be possible?

 

 

Alchemic



#4 mdfenko

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Posted 12 March 2015 - 03:48 AM

do you see the free peptide on the gel?

 

i just did the math and your problem may be based on the mw of the peptide and rna. the rna should be about 132 kDa and the peptide should be less than 1.5 kDa. you probably won't be able to see a significant difference in mobility between the rna and the complex with a 4% gel with so little crosslinking. you're looking for the equivalent of 4 to 5 nucleotides difference out of 400.

 

you might be to detect the difference with a capillary electrophoresis based sequencer.


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#5 alchemic

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Posted 12 March 2015 - 04:21 AM

Actually, I have a radiolabeled RNA and I observe only a radiolabeled signals of free RNA and RNA bound to protein. I have never observed a protein in a gel. Should I stain a gel with Ponceau to see a peptide?

 

Would greater crosslink of gel help with visualisation? Or maybe gel of higher percentage?



#6 alchemic

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Posted 12 March 2015 - 04:34 AM

I forgot to ask:

how to convert RNA size to kDA ?

or kDa of peptide to nucleotides?



#7 mdfenko

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Posted 12 March 2015 - 05:18 AM

you may be able to separate the difference in weight by increasing your monomer/crosslinker ratio to 29:1 (that's the ratio used with sequencing gels). 4% may be okay but, if not then try 6%.

 

for the conversion:

 

a single nucleotide is ~330 Da (this may be for deoxy nucleotides, then for rna it would be even more, by about 16 Da, it's still okay for a ballpark estimate)

 

a (very) rough estimate of the average amino acid is ~110 Da


Edited by mdfenko, 12 March 2015 - 05:19 AM.

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#8 alchemic

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Posted 12 March 2015 - 05:34 AM

mdfenko,

 

thanks for the advice.

I will try with 29:1 gels or 19:1.

 

Your help is invaluable. Thanks again.






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