rayh at fcspress.com
Sat Nov 5 05:34:59 EST 2005
Send a thief to catch a thief: try using goat serum to block goat non-specific binding - ideally it will contain proteins with the same reactivity as the 2nd layer and should block its binding. Using mouse serum alone will probably worsen the case (mouse serum may bind, then its binding will be amplified by the 2nd layer, so you could end up with direct and indirect non-specific binding) - this may better reflect the actual background binding in your experimental staining of course:).
I'd suggest that you use goat-blocked GaM-PE-stained cells to see if you can block the secondary, and introduce mouse serum (or an isotype control antibody) after blocking with goat serum, followed by GaM-PE, so that you can tell if the goat-blocking blocks the mouse too. If the blocking works, use it for your experimental staining as well as the control. If the goat-block doesn't stop mouse background, it might be worth trying FAb2 or Fab fragments of your mouse reagent, or trying to get directly conjugated mouse reagent so that you can block it more easily.
----- Original Message -----
From: Mark D'Amico
To: Cytometry Mailing List
Sent: Friday, November 04, 2005 4:03 PM
Subject: Best blocker?
I am using a secondary (goat anti-mouse IgG-PE). I am running the secondary with the cells (human) alone as one of my controls and get a bit more binding than I'd like. The incubation takes place in 2% serum ( I have used bovine and human) with similar results. Should I try mouse serum for the secondary incubations only?
Mark D'Amico, Ph.D.
207 Perry Parkway #2
Gaithersburg, MD 20877
(240) 243-8000 Ext. 206
mdamico at panaceapharma.com
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