Protein disappeared after TCA precipitation - (May/30/2006 )
I have precipitated some protein samples with TCA acid and ressuspended them in gel loading buffer (glycerol, tris, edta, sds and bromophenol blue). The samples that I ressuspended turned out yellow instead of blue!
Then I added a NaOH until the samples turned blue again.
Well, the problem is that after the electrophoresis my proteins dissapered (there are no proteins in the gel).
Can anyone tell me why this happened??
either your precipitation didn't precipitate enough proteins
or you had add too much NaOH leading to a very alkaline pH, ...but time was short between NaOH and loading on the gel? if not, maybe you digested your protein?
thaks for your reply.
Maybe you are right. Is there any other way in witch I can avoid losing my protein sample, the next time?
I also noticed that the proteins in the lanes next to this lane were distorted! I am not realy sure it was because of the high NaOH added...
the sample turned yellow because of the acid (tca) present. you neutralized with naoh turning the bromphenol blue back to blue but you added too much naoh and it hydrolyzed your protein. we neutralize by the addition of a few ul of 0.5M tris pH8 (until it just turns blue again). this is gentler so it won't hurt your protein (it may hurt your stacking if you add too much).
hi BioPe ! you can use TCA diluted in acetone, it works well. acetone removes most of the TCA and you are left with very little TCA in the sample.
After TCA precipitation, I wash pellet twice with cold acetone.
no problem of pH. No need to add NaOH
ThankS for the advices, Missele and SHIVA KESHAVA !!
Have you washed with acetone before putting loading bufer in? maybe you didn't dry enough and the acetone residue in the pellet is "running out" of the gel and your proteins get disstorted. In my case, it was an excess of salt that made all the proteins look like in your case. But that happened while I was using TCA, and not acetone precipitation, with acetone precipitation you have to dialyse the samples before thinking of putting them into 2X SDS loading buffer.