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Pulldown with nickel beads?

eluting from nickel bead

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3 replies to this topic

#1 rkay447

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Posted 30 July 2013 - 01:01 PM

I am hoping for some advice on doing a pulldown from yeast cell lysates expressing a Hex-HIS tagged protein.  My boss thinks I should be able to just boil the nickel beads in SDS-loading buffer but I'm not sure if this will work.  Has anyone done this?



#2 Curtis

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Posted 01 August 2013 - 10:07 PM

Weren't you the person who told me to boil my protein G in SDS-loading buffer at the end of IP?



#3 rkay447

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Posted 02 August 2013 - 07:48 AM

Hey there Curtis!  Long time since we've talked.  I hope all is going well for you.  Yes, a normal IP that uses protein G to bind antibody is eluted from beads using SDS and boiling but I'm wondering about nickel beads.  There's no antibody and the poly-his-tagged protein binds to the metal ions.  Usually people do this when they are purifying protein so they elute off the beads using imidazole and competitive binding.  I just want to detect the proteins that are binding my poly-his tagged protein by western blotting and was wondering if boiling these beads in sds-loading buffer would elute the proteins from the bead.



#4 labtastic

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Posted 30 August 2013 - 04:31 AM

For nickel pull downs, we just elute with 300mM imidazole. Although it's usually good to avoid high salt in your SDS-PAGE samples, this much imidazole usually isn't a problem. If it is a problem, just acetone precipitate your elution sample to remove the salt (4 parts cold acetone, 1 part sample, incubate at -20C for 30min, spin at 16k x g at 4C for 10min, aspirate supernatant, dry pellet, and solubilize pellet in 1x SDS sample buffer). Another option would be to elute with 100mM EDTA. 

 

Hope that helps.






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