large protein - (Oct/23/2011 )
Hi There,
I'm trying to detect ATM (460 Kd) from Arabidopsis. I think I managed the first time but the WB signal was really high in the membrane. When I try to run it longer, I get all lower bands (I use HiMark ladder, Invitrigen) smearish in the gel. I ran it for about 5 h in the cold room but I'm not sure if it's wise because of the precipitation of the SDS, and still have the lower bands smeared like hell. The question is, can I run it O.N in 4oC in low current. Is there any other advise? (we use Tris-HCl biorad, 4%stacker;6%resolving).
Thanx a lot,
Guy
Presuming you actually mean tris-glycine (rather than tris-HCl), have you tried other gel types such as tris-tricine? I would not use a stacker, you probably want as much of the gel as you possibly can get, to allow decent resolution. Try running a longer gel.
With a 6% gel, you won't get any resolution of bands below about 70 kDa, and if you aren't interested in them, don't worry about them smearing/not resolving.
460 kDa is huge, you will probably struggle to get good gels of it, even 6% is probably too high a percentage.
You should be able to run it in the cold room with no problems, the SDS shouldn't precipitate while running, as the current will generate heat. You will need to have SDS and methanol in the transfer buffer, and you would be well advised to try different membrane types.
if you want to ensure that the detergent doesn't crystallize in the cold then you can use lithium dodecyl sulfate instead of sds. it's a little more expensive but it's soluble in the cold.
you may also want to reduce your acrylamide to 5% (you could also reduce the percentage of crosslinker).
if you want to sharpen the lower molecular weight smear then you could run a gradient gel.
Hi guys,
Thanx a lot, we'll start with the pre-cast Tris-acetate 3-8% Invitrogen gels and see how it goes.
Cheers,
Guy