Immunofluorescence-sensitivity

[Guy Oshiro]

In article <33d360fd.23891111 at news.univie.ac.at>
a8803349 at unet.univie.ac.at (Martin Offterdinger) writes:

> I am currently using an IF system with an unlabelled primary antibody
> and a fluorochrome(oregon green) labelled sec. AB. I have to make this
> system more sensitive as I am only able to detect an overexpressed
> membrane receptor. My  questions are: Would a system like prim.
> unlabelled, followed by sec. biotinylated  AB followed by fluorochrome
> labelled Avidin be more sensitive than the system I am using now?
> Would an expected  increase of a factor 10 in sensitivity be
> realistic?

I think that you can expect about a 4-fold increase in sensitivity by
the above approach.  Another approach that you should look into is the
use of a fluorescent substrate for an enzyme linked reaction.  Amersham
sells an ECF kit (enhanced Chemi Fluorescence) that basically couples
Alk Phos to the secondary and the AP acts on a fluorescence substrate
(called AttoPhos).  I am planning to test that particular system out in
our lab this week - so I don't have any real data to share with you.

Good luck

Guy Oshiro
EMAIL: Guy.Oshiro at UCHSC.edu

  /\  /\    /\
 /  \/  \/\/  \ Science in the Rockies.
/    \   \/    \