Nuclear staining instead of cytoskeleton - (Aug/12/2006 )
QUOTE (Casper @ Aug 13 2006, 05:17 PM)
How does your DIC look?? Do the cells maintain theri integrity???
I think you might want to try your fixing with PF and cold methanol. Sometimes trition can polymerize actin/tubulin filaments. So what you might be actually seeing as lumps in ur nucleus might be clumps of cytosolic or cell membrane fragments.
I think you might want to try your fixing with PF and cold methanol. Sometimes trition can polymerize actin/tubulin filaments. So what you might be actually seeing as lumps in ur nucleus might be clumps of cytosolic or cell membrane fragments.
Thank you Casper for this info, I did not know that triton could have that effect on actin/tubulin filaments. But why would I see those clumps only in the nucleus if they are cytosolic or if they are membrane fragments? I see them in every nucleus from my cell culture.
-Edwige-
Is the presence/absence of the NLS actually such a hard & fast rule? If it looks like its in the nucleus, I'd be inclined to say its in the nucleus, myself.
Have you ruled out cross reaction of your antibody with some other (nuclear) protein? Can you get another antibody to the same protein from somewhere else, or are you stuck with just the one. I'd try the WB if you have enough of the antibody.
Best of luck.
-chemist_guy-
QUOTE (chemist_guy @ Aug 31 2006, 06:12 PM)
Is the presence/absence of the NLS actually such a hard & fast rule? If it looks like its in the nucleus, I'd be inclined to say its in the nucleus, myself.
Have you ruled out cross reaction of your antibody with some other (nuclear) protein? Can you get another antibody to the same protein from somewhere else, or are you stuck with just the one. I'd try the WB if you have enough of the antibody.
Best of luck.
Have you ruled out cross reaction of your antibody with some other (nuclear) protein? Can you get another antibody to the same protein from somewhere else, or are you stuck with just the one. I'd try the WB if you have enough of the antibody.
Best of luck.
I think chemist guy's suggestion is the best if there's another antibody. A Western is a waste of time in my opinion at this juncture, particularly if you are having cross reactivity issues.
I have done a lot of F-actin staining always w/Triton X-100. Never a problem with epithelial cells.
Contact the lab you got the Ab from.
-viper-