I am trying to do His pull-down to search for interacting proteins of my protein, which is tagged with Myc His at the C terminal end. I have cloned my protein in the pCDNA 3.1 vector and transfecting the construct to HEK 293T cells followed by pull down with Ni NTA columns.
However I am getting lots of non specific bands in the vector transfected cells themselves. Do you have any suggestions as to how to remedy this?
His-tag pull-downs from mammalian cell lysates are fairly trickly. Firstly, there are polyhistidine containing proteins that bind in a specific fashion. Additionally, depending on your lysate conditions, you may get various type of non-specific interactions.
Since you have a Myc tag, you might be better off using 9E10 mAb to immunoprecipitate your target protein. The background from epitope tag pull-downs, esp w/Myc tags, is fairly low in my experience.
If you want to stick with the his-tag approach, you'll have to pay special attention to your cell lysis buffer. EDTA and SDS are commonly used reagents in cell lysis buffers that will interfere with NTA resins. Likewise, nucleic acids can be problematic (polyanions will bind nickel-NTA). Once you have conditions where your protein of interest does show bead binding, I would recommend extensive washing of your pelleted beads with buffers that contain low levels of imidazole, modest levels of non-ionic detergent, and increasing stringency of ionic strength, to strip off weakly bound or non-specific adsorbed proteins. Sometimes it is also helpful to pre-block your NTA-resin using BLOTTO (non-fat dry milk).
The background on the resin will never be zero, so no matter what, you'll have to run SDS-PAGE to evaluate your bound fraction, and assess for proteins uniquely enriched in the bead fraction isolated from your transfected vs. non-transfected lysates. Some level of non-specific background can be tolerated so long as you can still resolve bands of interest.
Thanku very much Daniel.. You are so nice.. actually I need to use this His Pull down only...we are running out of the myc antibody...
The Iowa Hybridoma Bank sells the 9E10 antibody at low cost,
http://dshb.biology.uiowa.edu/c-myc
although I don't know if they ship overseas.
You will probably save time/money in the long run with the Myc IP approach.
Cheapest way is to add increasing amount of imidazole in your washing steps to get rid of impurities. Be careful not to lose your protein of interest. Another way (more expensive) is to use HisPur Cobalt Resin (Thermo) which are much more specific and result in lower background.
I agree with this. You will probably be able to tell apart your proteing from the rest adding a gradient with increasing concentrations of imidazole (like 50, 100, 150 and 250 mM)
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