The firsr WB (anti-GST), no bands? that is not correct. even thrombin
cleaved may contaminated with some GST
The 2nd Wb. (anti-nef), see both GST and nef bands. Then question here: what
is the molecular weight of the bands? GST should be around 25 Kd, GST-nef
should be > 25 Kd.
What is the results of 3rd Wb?
If u thougt anti-nef would cross react with GST, just add excess amount of
GST in your anti-nef solution to adsorb the non-specific antibodies and see
what is the result.
please visit our web http://www.immunechem.com for our antibodies and
glutathione agarose products.
svt14 wrote in message <364348DE.41C6 at cict.fr>...
>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
> -after thrombin action we eluted gst by competition with reduced
>>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
>>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!!