2 replies to this topic

[mdnworms]

Hello, I was hoping someone could help me with some Gel shift/supershift issues.  I am using the Pierce Biotin EMSA kit, that many here have used.  So what I have done so far, is 6X-his tag purify my protein, which shows some non-specific proteins of different sizes coming out with the purification (See Gel Picture). I used the Qiaexpressionist protocols.  

I was told by some to just try the gel shift anyway, and see what I get.  So I followed the kits protocol exactly, and I also supershifted using an anti-histag Ab (See Gel Shift).  Everything appeared to work nicely (Lanes 4-8) except the contaminating proteins bind to my probe as well as the kits probe and cause non-specific shifts (Lane 2).  To make things more confusing the anti-His tag Ab caused a supershift with the kit's probe (Lane 3)??  However when I mutate the binding site in my specific probe I eliminate the large band that I think is my protein, but still see a super shift (Lanes 9 and 10)??I have attached a picture of the membrane.  Does anybody have any suggestions?  Should I be including blocking agents (BSA or detergents), in my incubations?  Is my protein just sticky??  Why would my protein shift the kit's probe?? Very strange.  Thanks, I would really appreciate any input!!!!

M

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Edited by mdnworms, 10 June 2009 - 12:54 PM.

[mikew]

Hi,

#1. Your purified protein binds the probe that comes with the kit (whatever that is).
This indicates that your protein will bind any probe non-specifically.
#2. The results of the binding with your probe and the mutated probe looks reasonable.
If you leave out lane 10 the lanes from 4-9 would look logical. Your protein shifts with the antibody and the mutated probe doesn't bind.
  However, you do have lane 10. So, it appears that the His-Antibody is complexing with some protein in your mix
to form a stable protein-DNA complex. This complex seems to form with any probe.
It would be a good idea to include BSA and detergent (NP-40 or Chaps) in your EMSA mix.
This may make the binding of the protein somewhat more specific.

[mdnworms]

Thanks for the input.  Yeah, I was kind of worried that my protein was just sticky and would bind to anything, but there is that mutant data??.  I looked at the Kit's probe sequence and there are some stretches that resemble but are not identical to my binding motif, so maybe that is responsible for the shifting I see there.  Needless, to say I am going to make a better negative control, and try the BSA, and NP-40.

Do you have any suggested concentrations?  I have already done what the kit suggested: 0.05% NP-40, but have not tried BSA or CHAPS.

Also, any suggestions with salt concentrations (ie. KCl, MgCl2, etc.)?

Appreciate the help!