Renee Lapointe wrote:
> 1) I would like to get some information about the use of anti-GFP
> antibodies to augment the GFP signal in fluorescent microscopy. We have
> been using EGFP as a reporter for gene expression from various promoters,
> but it seems that EGFP needs to accumulate in the cells for a long time
> before the signal is detectable (24 hours). We are interested in knowing
> the % of cells which are expressing from these promoters regardless of the
> level of expression. Therefore we would like to know whether the use of a
> GFP specific antibody could improve the signal. Has anyone tested that?
>> 2) I guess this brings me to the second part of my message. CLONTECH, the
> company which sells these vectors, also sells a polyclonal antibody against
> GFP, but it's titer for immunofluorescence is 1/200, rendering the cost of
> its use too high for us to use (we have numerous promoters and conditions
> to test). Does anyone knows of a cheaper source of GFP-specific antibody
> which would detect EGFP even in a denatured form (for westerns for example)?
>> Thank you for your help,
>
Hi Renee,
sounds funny, but as a test of staining conditions I have done
fluorescent anti-GFP stainings of Drosophila brain neurons. I used
Molecular Probes polyclonal Rabbit anti-GFP (1:1000) with a Cy3-labeled
secondary antibody.
I didn´t quantify the results, but the images looked very bright and had
a high contrast. So I´d say it is worth a try.
Thomas Siegmund
Institut fuer Genetik
Freie Universitaet Berlin
