Burned membrane?.... - (Aug/23/2006 )
I need your advice....
I did an IP using Flag tagged beads against my Flag taged protein.
After transfer, I use poenco to visualize the band and I saw the heavy chain and my Flag-Protein.
then I wanted to check for phosphorylation (of my protein...) and I used a specific antibody (Rabbit origin). Out of the 4 lanes I got a very intensive signal in the lane where I expected my protein to be phosphorylated.... till this moment I was very happy.... But-
I stripped the membrane, blocked again and then incubated with M-anti- Flag antibody ( I want to see my protein in all the 4 lanes), but I got a signal from all of the lanes- except from the one that was exposed to the R-anti-phospho antibody.... I thougt that the band is suturated with the rabitt Ab so I used a more harsh stripping- but still the same.
A freind told me that somtimes when membrane are exposed with a very intense and high signal, they are getting burned in this spot.....
But I can't understand how can it be, or what is the explanation for it?
I would like to know what other people think....
I've never heard about burned membrane
did you check your stripping ? (incubate in blocking, add ECL subrate and expose a film : if there is a signal, the second antibody is still there), if no signal, incubate with second antibody and ECL substrate and film exposure, : if there is a signal, the primary antibody is still bond)