[Histonet] Re: Casein block for phosphorylated antigens?

[Johnson, Teri]

I found this from a Google Search -
http://www.nanotools.de/protocols/western_blot.php

"2. Blocking of Nonspecific Binding Sites

    * Block blots for at least 2 hours with Blocking Solution. Blocking
overnight is also possible (add phosphatase-inhibitors, e.g.
sodiumfluoride, sodiumvanadate if the assay targets are phosphorylated
epitopes).
    * Wash three times 5 min with 2x PBS/0.1% (v/v) Tween 20.

General Blocking Solution
We recommend a casein/Tween 20 based blocking solution (e.g. nanoTools
product #3031-500/CPPT or #3031-3000/CPPT) for the use with
phosphorylation-site specific antibodies and antibodies that are not
directed to phosphorylated epitopes, unless otherwise stated in the
datasheet.

Alternative Blocking Solution
This blocking solution is recommended for the use with
phosphorylation-site specific antibodies that cannot be used with a
casein based blocking solution. These are especially the
phosphoserine/threonine-specific antibodies that are included in our
Phosphoserine and Phosphothreonine Detection Kits (see list below).
Phosphoserine-specific antibodies: PSER-1C8; -4A3; -4A9; -4H4; -7F12;
-16B4
Phosphothreonine-specific antibodies: PTHR-1E11; -4D11; -14B3
For these antibodies, it is essential to avoid the use of blocking
reagents containing phosphoproteins (e.g. milk or casein), since this
will lead to competition of the highly phosphorylated casein with the
phosphoserine-/ phosphothreonine-specific monoclonal antibodies.
Application of casein or milk may lead to significant decrease or loss
of signal intensity due to reaction of the primary antibody with the
blocking/diluting agent. We therefore recommend to replace casein by 2%
(w/v) BSA."

This is on western blots. Does this also pertain to
immunohistochemistry? Please, freely discuss opinions here!

Teri

>  -----Original Message-----
> From: 	Johnson, Teri  
> Sent:	Wednesday, May 09, 2007 12:46 PM
> To:	'histonet <@t> lists.utsouthwestern.edu'
> Subject:	Casein block for phosphorylated antigens?
> 
> Has anybody heard of (or can cite references for) not using casein to
> block in IHC when immunostaining for phosphorylated proteins?
> 
> Teri Johnson, HT(ASCP)QIHC
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, MO 64110
>