RE: A poor man's Zenon.

Hi Uriel,

We use more Zenon than I like to tally.

I recall we've done a simple (primary+secundary goat F(ab)2 ) pre-incubation
experiment alongside Zenon and "as it should be done" primary, then
secundary staining experiment as the mixing experiment, already quite a
while ago now. Results of the pre-mix were awful, unlike the normal two-step
protocol using the same reagents or the Zenon single-step staining.

My guess is that the trick is to use monovalent rather than bivalent
secundary for Zenon-style pre-incubations. As I haven't found a non-Mol
Probes provider of directly labelled IgG Fc specific Fab's (or looked very
hard, I admit), I continue to rely on Mol Probes.

Hope this helps,

Guy

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-----Original Message-----
From: Uriel TK [mailto:utk1@013.net] 
Sent: Tuesday, February 22, 2005 11:15 PM
To: cyto-inbox
Subject: A poor man's Zenon.

Hello friends!

I was looking at mol. probe's Zenon stuff, and I was wondering whether that 
could be possible with normal 2ry Abs. If one was to use a purified primary 
antibody and a 2ry one (F(ab')2 or F(ab) from jackson for example), 
shouldn't it be possible to achieve the same as probe's product? All you 
need extra is "stock" IgG or a purified monoclonal to block the reaction. It

would go as follows:

1)titrate your 1ry and 2ry as you'd normally do for a 2 step labeling (100 
microL reaction vol)

2)Prepare 6 tubes with the amount of 1ry and 2ry determined in "1" but in a 
50 microL volume, incubate, and then add 50 microL of serial dilutions of 
blocking IgG (to bind the left free 2ry).

3)label 6 samples of your cells with a different 1ry of the same isotype, 
wash, and then do a "2nd step" consisting of the 100 microL mixture obtained

in "2".

4)run an experiment with unstained,  normal 2ble stain, blocking IgG+2ry Ab 
(control for unspecific staining) and the 6 samples obtained in "3".

If the amount of blocking IgG is too low, I'd expect to see an increase in 
fluorescence (in comparison to the normal stain) due to excess 2ry binding 
the "other" primary Ab. But if the blocking is good then the fluorescence 
should be quite close to the "normal" 2ble stain + extra background from 
unspecific IgG-2ry binding, which shouldn't be much. The molar ratios 
obtained should stay quite constant for all Abs of the same Fc and would be 
adjusted according to the amount of 1ry used. Thus you could prepare custom 
reagents with whatever color you have on your 2ry which is compatible with 
multiple labeling, even using primary Abs of the same host since the 1ry-2ry

interaction is done for every component separately.  This system would also 
save money and the QC of Jackson I understand is as good as mol. probe's. 
Instead of buying multiple conjugations of the same marker (or paying a lot 
for zenon) you only need the purified and you can pick your colors according

to every experiment as needed and need not to worry about host species. This

seems so simple that there must be a caveat somewhere, surely someone tried 
it in the past.

I would very much like to hear (read) your comments on the subject.
Thanks in advance,
Regards,
Uriel. 





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