Problem with TCA and western blot - (Jun/02/2005 )

Hi ALL,

I have a problem with my western blots which I think is bieing caused in soem way by TCA precipitation. I am precipitating scFv protieins from MRS meduim using 10% TCA, The pellets are then washed (70% EtoH) and the run on SDS-PAGE before western bloting, The SDS-page gels run fine but my tranfer is a mess with alot of smearing. It I don't TCA precipitate my proteins the transfer is fine.
Does anyone have any suggestions as to how I can get my westerns to work properly?

hi
when i precipitate proteins using TCA method, i add Acetone too
1vol sample, 8vol acetone and 1 vol of TCA (add this order)
pellet and wash by with acetone adding 4x initial vol.
pellet and dry
resupend in sterile ddH2O

The problem may be either due to your sample remaining too acidic or the proteins not redissolving. Does your loading buffer turn yellow? Do you boil your samples before loading?
P.S. I agree with the above about including an acetone wash.

Hi

I precipitate yeast proteins with 20% TCA, then beat them with beads to smash open the cells. I then transfer the proteins and debris into a new tube. After which, I spin everything down, remove supernatant and wash the pellet 3 times with 5% TCA. Finally I resuspend in sample buffer


10% b-mercaptoethanol
300mM tris pH 8.8
4%SDS
20% glycerol
.0025% BPB

My samples run fine on gels, better than when prepared with a lysis buffer

Pat