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Immunofluoroscence (IF) staining problem


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5 replies to this topic

#1 hmohamma

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Posted 09 March 2005 - 08:45 PM

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

#2 Cyanide

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Posted 16 March 2005 - 02:34 PM

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.

#3 joy_maf

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Posted 20 March 2005 - 12:47 PM

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.

<{POST_SNAPBACK}>



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

#4 hmohamma

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Posted 20 March 2005 - 03:24 PM

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




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.

<{POST_SNAPBACK}>



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

<{POST_SNAPBACK}>



#5 Scott

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Posted 21 March 2005 - 05:16 AM

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

#6 joy_maf

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Posted 25 March 2005 - 01:59 PM

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




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