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?
Pulldown with nickel beads?eluting from nickel bead
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?
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.
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.