antibody for immunocytochemistry - only validated for Western-will it work? (Apr/08/2009 )
Does anyone know if I can use an antibody that the data sheet specifies is "only validated for Western blot" to stain cells grown on chamber slides? Thanks!
There is no way of knowing whether or not an antibody that hasn't been tested for ICC will work until you try. I always test antibodies at varying concentrations to see if it will work. You may also want to try a couple different cell lines since it may or may not be specific to a species. Just be sure to also include an IgG control to determine if any staining you do see is background or specific.
...and start at a high concentration. Most Abs can be used for Westerns at a concentration 2-10 times less than they can for immunofluorescence.
As to the species, if you're using an Ab that was raised against an antigen of a different species you can always do a protein alignment (using ensembl or similar) and see how high the homology is between the species the antigen was taken from and the species of your tissue / cells. I worked in zebrafish and no one makes anti-fish abs, but there are plenty of other abs out there that do just fine.
Also, do a literature search for your antibody and see if it's used for immunofluorescence in the literature at all. Chances are you won't find it because they probably would have referenced it on the data sheet, but you never know.
Thanks for these replies! Most helpful.
I dont think that it will work coz the binding site of th antibody is not the same for folded (immunocytochemistry)
and unfolded (western after denaturation)