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blocking the resin for his-tag pull down


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#1 Denis Chaix

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Posted 01 November 2010 - 02:30 PM

Hello everybody

I'm trying to do a pull down experiment with a His-Tag peptide.
I use 100 uL of NiNTA resin and I add 10 ug of peptide.
Incubate during 25 min.
wash 1 time with 500 uL of buffer A ( 20 mM hepes pH8, 100 mM KCL, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM imidazole).
I put the eggs extract and incubate 30 min.
after that two washes (1 mL) with buffer A.
Elution with buffer A + 300 mM imidazole.
But the problem i have to much protein in my elution specific or non specific.
I want to know if some one have an idea of blocking protein or reagents to saturate the resin after the addition of peptide to reduce the non specific binding like in a WB experiment.

thank you

#2 protolder

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Posted 01 November 2010 - 11:13 PM

Hola, I canīt answer your question, but with 100 ul of resin you have capacity of capture around 1 mg. of 6His protein. THe best way to decrease inespecific interactions is lowering resin volume and add 10-20 mM imidazole to the eggs extract. Good luck

#3 Denis Chaix

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Posted 02 November 2010 - 06:27 AM

In fact I have already try increase the concentration of imidazole until 30 mM in eggs extract.
I will try to decrease the volume of resin.
Thanks for the reply

#4 Denis Chaix

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Posted 02 November 2010 - 05:06 PM

Any other other ideas ?????




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