western blot - western blot (May/27/2009 )

dear all

do u think is possible to see in western blot in the same lane 2 proteins with 4 KDa of difference?
one of 220 KDa and the pther form of 216KDa????

I would say it's possible, I already saw such result but you need to add glycerol in the gel. Never did it myself. I have no protocol. sorry

I think it might be, if your gel is an high acrylammide-bis acrylammide % gel (15% or more)

it's difficult, but if you run the gel long enough and, as Velella suggested, use a high % gel it should be possible in principle.

people do similar things for ubiquitin which is 8.5 kD

I have been able to see a 4kb difference when running a gradient gel at lower speeds (For longer time). 4-20% gradient run at 80V (regular 5cmX7cm gel).

we separated proteins in the range you need (isoforms of myosin heavy chains) by running a 3.5-5% gradient gel with a 8-0M urea gradient.

we ran 3.5-5% acrylamide with a 8-0M urea gradient gel (neville buffer formulation, not laemmli but that may work, as well) to separate myosin heavy chain isoforms. it should work for you.

this is the third time i am seeing the same question. you only need to post the question once. more than that becomes annoying.

as i answered twice before, a 3.5-5% acrylamide with 8-0M urea gradient, sds-page, was used to separate myosin heavy chain isoforms. try it if you wish.

mdfenko is not going nuts -- neither are you. I merged what was originally three threads asking the same question into one thread. mdfenko answered the question as posted by distazio in all three threads, and since the answers were not identical, I didn't feel I should delete any of them...

tnks

i'll try like mdfenko suggests.