Which side of the membrane should face up during incubation steps? - Does it matter? (May/28/2010 )
I know that in general, the protein side of a membrane is placed face UP in the incubation vessel during the blotting portion of the Western blot protocol. However, can you incubate the membrane protein side face DOWN? Does it matter? Is one better than the other?
I understand that the membrane should definitely be faced up at the very end to detect signals.
if you put the membrane into the tray face down then you have to ensure that it doesn't stick to the bottom. otherwise you will get uneven exposure of the face side with the solutions.
bottom line is the protein on the blot shud be able to take up the antibodies!!! anyside u place it does not matter... but the blot facing down makes it more difficult for even binding of antibodies as md says... moreover to avoid tat u might need more volume of antibody solution.
So the best deal is to make it face u so tat u ease the pratical handling aspect of it!!!
It doesn't matter so long as the protein-bound side can make contact with the antibodies. it also doesn't matter in which way you place it on the rocker. I had another post-doc once tell me that my membrane HAD to be rocked in landscape (not portrait) orientation!
than4 on May 31 2010, 06:28 AM said:
why is orientation of shaking important? We have both types of shakers standing next to each other and depending on my mood i'm using one day this and the other day the other shaker...i noticed no differences