Immunofluoroscence (IF) staining problem - problem in NF-kB staining (Mar/09/2005 )

Hi everbody;

I am doing IF staining of NF-kB. I do not know what is pattern of its localization in the cell. I was wondering if there is anybody to know that or guid me to get some pictures about that. The secong subject is that IU am looking for a good activator of NF-kB. I have got the NF-kB gene as well as HIV Tat gene. But I do not know how I can see nuclear translocation of NF-kB induced by Tat and how long aftertransfection.

I do appreciate your help in advance

Haki

-hmohamma-

Hi, I'm also looking at the translocation of NFKB you can get a kit or carry out a western blot or an electrophoretic mobility shift assay. However at present I can not get it to work!! I think I'm having problems with my nuclear extracts.

-Cyanide-

QUOTE (Cyanide @ Mar 16 2005, 03:34 PM)

Do a Ikb degradation time course first to determine the time window for NF-kb translocation (IF, Western or Nf-kb reporter assay.

-joy_maf-

Thanks for your comments. Would you please tell me what it could be a positive control for NF-kB translocation by IF?

Thanks alot

Haki

QUOTE (joy_maf @ Mar 20 2005, 01:47 PM)

QUOTE (Cyanide @ Mar 16 2005, 03:34 PM)

Do a Ikb degradation time course first to determine the time window for NF-kb translocation (IF, Western or Nf-kb reporter assay.

-hmohamma-

Hi Haki,

Quite a few years ago I was looking at nuclear translocation of NFkB in various cell types using IF. I found that staining with a p65 antibody from Santa Cruz targeted to the nuclear localisation signal followed by a tagged secondary gave a good visualisation of translocation in response to LPS. From memory it was at 6 hours I was looking.

Hope this helps

Scott

-Scott-

TNF-a, LPS, IL-1b, ROS (H2O2), integrin Ab......
First, do a time couse study on your cells using the stimulus you pick.

-joy_maf-