The goal is to detect HIV-1 Gag and Env proteins being expressed
from a plasmid which is being transfected into HeLa and HeLa-pTev
(stably expresses HIV-1 Tev protein) cell lines. The method of
detection being used is western blotting and enhanced
chemoluminescence (ECL; [Amersham]). The primary Ab used is a
pooled sera from HIV-infected patients. The secondary Ab is
anti-Human IgG-POD (Boehringer-Mannheim).
The human sera appears to be picking up HeLa cellular proteins in
general. This has been observed with plain HeLa cells, as well,
when they are used as negative controls. There is no background
on the nitrocellulose membrane itself. The membrane is blocked
prior to Ab incubation with 5% dried milk in PBS-1% Tween20. Ab
have been incubated using PBS-Tween20 alone and with 5% dried
milk. Results were the same with both. Different Ab titres have
been tried as well. Also, there is no evidence that the proteins
of interest are being detected at all or they at least don't show
up through the background. We know that they are there because
p24 ELISA's are positive and mRNA has been detected by RT-PCR.
The positive control consists of lysate from HIV-1-infected MT4
cells. For these, when run in tandem with the above test samples,
gives no non-specific background and the HIV-1 p24 and p17
proteins are clearly evident.
1. A suggestion that was given was to pre-adsorb the sera with
HeLa protein lysate. Aside from the protocol in Maniatis, are
there any other protocols that you have tried? Our preliminary
results are not promissing.
2. We have p24-, p17, and gp120-specific Ab (from NIH through
NIAID), however, the secondary Ab is on backorder for one month.
Has anyone used Ab from NIH with any success? We have heard that
they aren't that great. I can give Cat. numbers if necessary.
3. Personnel at the Ontario Ministry of Health HIV lab have told
us that the detection of gp120 is practically impossible with
western blotting unless the protein is actually purified. Our
lab's past experience was not promissing. Any
experience/suggestions on this or on how to detect Env proteins.
We are currently trying immunoflourescence with the above gp120
All replies will be greatly appreciated and credit will be given
Dept. of Microbiology
U. of Toronto
r.lombardi at utoronto.ca