Dear Mari

We are using the Jackson Cy3 antibodies since many years with excellent
results.  We prefer much higher concentrations than you applied (routinely
1:200).  I think your problems could be based on several reasons:  1.)
Perhaps the sensitivity of this secondary antibody is much better than the
previous, and you have to reduce the concentration of the primary antibody,
because that's the trouble maker.  2.)  The antibodies sometimes tend to
precipitate (see Martin Wessendorf's message).  A simple solution is
spinning the antibody at full speed in a microfuge for 5' and transfer
supernatant to new tube.  3.) The blocking could be wrong.  In our
experience, best results were obtained by incubating the antibody for 30' at
15°C in PBS containing 2 - 10% of normal serum from the same species in
which the secondary antibody was made (plus optional 0.2% Triton).  4.)
Check whether you are using the correct filter sets.  You should also keep
in mind that what counts is the signal to background ratio, rather than the
absolute background.

Particularly the donkey Cy3-conjugated secondaries from Jackson are
excellent.  You shouldn't have any persisting problems (unless something is
wrong with your lot).  If problems persist feel free to send your detailed
protocol and I will check for other possible problems.

Good luck,
Jacques

---------------------------------------------------------------
Jacques Paysan, PhD
University of Hohenheim
Institute of Physiology 230
Garbenstrasse 30
D-70593 Stuttgart
Germany

Phone: ++49-711-459.22.67
Fax:  ++49-711-459.37.26
email:  [log in to unmask]
Web:  http://www.paysan.de/lynx.htm


----- Original Message -----
From: "Shinohara, Mari" <[log in to unmask]>
Newsgroups: bit.listserv.confocal
To: <[log in to unmask]>
Sent: Friday, August 11, 2000 4:57 PM
Subject: questions on Cy3


> Hi,
>
> I am now trying to see subcellular localization of molecules.  When I use
> FITC as secondary staining, it looks nice.  However, if I use Cy3, there
> appears pretty high background (all over, not only in cells) and the whole
> field looks orangish as well.
>
> My questions are...
> 1. Am I using too much Cy3?  (I use Jackson's SAv-Cy3 at 1;2,000 dilution.
> Stained for 30 min at 4C-degree)
> 2. Someone told, if I remember correctly, Cy3 is a rather big molecule.
If
> this is correct, can  intense washing get rid of the big molecules off
from
> cells and outer fields?
> 3. Is there any good red-color alternatives that gives less background?
>
> Any suggetion will be appreciated.
>
> Thank you.
> Mari