VERY STUPID QUESTION - (Oct/06/2005 )
Hi! Im running an electrophoretic transfer of some proteins, and then incubating my membrane is some solutions, but I am worried that when I placed my membrane in my solution, I did so upside down so that the proteins are face down. This would be bad, so does anyone know how to check if I did this? Is there a non-permanent stain I could use? Coomassie Blue perhaps??
thanks very much
Ponceau red will verify your protein transfer and washes off in tap water. Or, why not mark the membrane with a pencil so you know which is the protein side??
if dry milk for blocking was one of the solution Ponceau will not be washed so easly
BTW, I mark my membranes with a pencil, as was suggested by frustrated.
But, I'm curious why washing the membrane right-side down would be such a disaster. I don't know exactly the experiment you're doing, of course, but unless there's some good reason to be very concerned, I'd just soldier on, it's likely everything will be fine...
i have done that before and it's been OK (i notch the corner and sometimes I forget to note which way I notched it )
just use plenty of wash volume and you are fine
sometimes if you get to the end and your pic is a little fuzzy, turn the membrane upside down for a bit more clarity (if it was upside down)...but the light will go through the membrane so it really shouldn't matter as long as you are not a total miser with your buffers and incubation volumes
I agree, why would it be bad?...if you still know on which side you have the proteins there's no pb in washing the membrane upside down as long as some buffer gets under it. If you don't know where your proteins are for ex for ecl detection you can can do it on the other side if you don't have any signal. I have even made antibody incubation with 2 filters in one box to save antibody: one filter with proteins down and one filter with proteins up.