2 replies to this topic

[Julie123]

hello All,

I am transfecting a plasmid that expresses a flag fusion protein.the flag antibody is cross reacting with another protein that is bigger than the fusion protein.is flag 1 kda?what can i do to reduce this non-specificity?is it possible it is my secondary antibody(anti-rabbit 1:2000)?thx i appreciate your help here.

Edited by Julie123, 31 March 2010 - 08:16 PM.

[goldfinger]

Why bother? as long as the nonspecific band is not overlapping with your band.

I use 1:10,000 dilution of anti rabbit-HRP.

[Julie123]

goldfinger, on Apr 5 2010, 12:39 PM, said:

Why bother? as long as the nonspecific band is not overlapping with your band.

I use 1:10,000 dilution of anti rabbit-HRP.

Hello Enthusiat

the probelm is that i see a band even in cells that were not transfected with the vector that has flag+insert.I tried polyclonal antiflag from sigma and it is nonspecific.I also tried flag M2 and in this case i dont see any band and if i use anthor M2 anti-flag (different catalog nb)i see alot of nonspecific even in the nontransfected cells. I am not sure what is happening.Either antibodies are not good or my transfection didn't work well.Has anybody used lipofectamine to cotransfect siRNA and plasmid in Hela cells ?
Thx