[Histonet] phosphorylated antibodies

[Henry, Charlene]

Fellow Histonetters,
I was ask to respond to all about my experiences with phosphorylated
antibodies and am sorry it took so long to respond, but we have been
extremely busy. We currently have approximately 8 phosphorylated
antibodies that we are using for various research projects. Some of the
antibodies have been developed without any problems while others have
been a little more stressful. When we develop any antibody, we test the
antibody with at least 2 different detection kits (usually IVIEW, LSAB2,
LSAB+, Envision, and CSA if all others fail) and different pretreatment
buffers. With a few of the phosphorylated antibodies (pEGFR, pP70 S6
Kinase, and pRb) we had to use Dako's CSA detection kit to get them to
work. By using the CSA, you amplify background as well as the targeted
protein; so you have to use a lot of buffer rinses between your steps.
Since I was ask about the pEGFR in particular, we get our antibody from
Cell Signaling cat # 2231L and we use it at a 1:200 dilution with the
CSA II detection Kit  and the pretreatment is Proteinase K for 10
minutes. Since the antibody is rabbit polyclonal, you will need the CSA
Rabbit Link for the CSA II kit instead of the link included in the kit.
The pEGFR is the only one of the phosphorylated antibodies that we have
that works better for us with an enzyme pretreatment instead of HIER
pretreatment.
Hope this helps.

Charlene Henry, HT QIHC
Histology/IHC Section Head
Department of Pathology
St. Jude Children's Research Hospital
332 North Lauderdale
Memphis, TN  38105
Phone: 901-495-3349
FAX: 901-495-3100