IF - Methanol Vs. Triton? - Cytoskeleton and Membrane associated proteins (Jun/01/2008 )
Just an immunofluorescence question. I am looking at two different proteins, one is membrane associated and one is cytoskeletal associated. I have found through literature and practise that for the membrane associated I have to permeabalize using triton, and lose the epitope when I permeabalize with methanol, and vice versa for the cytoskeletal protein.
Does anyone know why this is??
Different fixation methods are designed to promote the preservation of specific cellular components. You can do some research on the internet and discover why each fixation is best for each application but there are specific fixation methods that can be used to preserve both membrane associated proteins as well as the cytoskeletal proteins. The most common is paraformaldehyde/glutaraldehyde fixation. Just search "immunofluorescent fixation protocols" and you can find lots of info and protocols.
Hi, have a glance on this: http://publish.uwo.ca/~jkiernan/formglut.htm
Maybe it can be useful to have an idea of what fixative is better for you. In my case, paraformaldehyde+glutaraldehyde is the best to fix my sample withouth altering the cell surface. Also, I use triton x-100 to permeabilize my sample when i want to label citoplasmic proteins. Good luck