incredible result on western blot!!

svt14 svt14 at cict.fr
Fri Nov 6 14:07:10 EST 1998


Hello immunoprofessionnal!
I'm a young french student. I need your help for an interpretation of
western data. Here is the experience : We have puirified three type of
protein on an agarose colon (agarose was coated to reduced glutathion): 
 -the complex gst-nef (hiv 1 protein) fixed on agarose-glutathion has
been eluted from the colon by competition with reduced glutathion
 -On an another colon Thrombin action allowed to separate nef from the
complex agarose-glutathion-gst
 -after thrombin action we eluted gst by competition with reduced
glutathion

These three proteins were studied by western blot.
1 Wb was revelated with an antybodie-peroxydase directed again another
antibody anti gst ===> NO revelation of ANY protein even the molecular
weight marker.
 
Th second wb was revealed with an Ab-pero directed against Ab anti nef
====> The ab revealed the complex gst-nef, protein nef until here all is
right but the gst was revealed too!!

The hypothesis that cannot be right is the mispurification of the
antibodies because the too solutions were preparated independantly
Can it be the thrombin that doesn't cut exactly between gst and nef an
let a piece of nef on gst ?? If you have other ideas about the two
western please tell me!! 

Please forgive me for my bad english!! It's my first essay!
I hope you'll answer me soon!!
Thankyouthankyouthankyou!!!!!!!!



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