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blocking the resin for his-tag pull down what kind of blocking reagents

blocking the resin for his-tag pull down what kind of blocking reagents - Protein Science

blocking the resin for his-tag pull down what kind of blocking reagents - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 11-01-2010, 10:33 PM
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Default blocking the resin for his-tag pull down what kind of blocking reagents



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
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Old 11-03-2010, 12:17 AM
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Default Re: blocking the resin for his-tag pull down what kind of blocking reagents

Any Ideas ????
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Old 02-16-2011, 04:00 PM
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Default Re: blocking the resin for his-tag pull down what kind of blocking reagents

First thing you should try is the simplest. Increase your salt concentration. 100mM is relatively low with NiNTA. Average is 250mM.
If that doesn't help in preventing non-specific interactions then you should look towards non-ionic detergents as they remove background proteins. Detergents such as Triton and Tween are highly compatible with NiNTA resin up to 2%, probably only need 0.2% or so though.
Take a look at the Qiagen website for a list of other compatible detergents.
Let me know if these ideas don't help.
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Old 02-17-2011, 06:44 AM
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Default Re: blocking the resin for his-tag pull down what kind of blocking reagents

Hola, with 100ul of resin you have capacity to bound about 4 mg of protein, so follow the above recommendations and lowering the resin to 10ul it is probably that your results improve. Buena suerte
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