We have a paper that is about to appear in CCC, describing activation of MEK and ERK using phosphospecific antibodies. We are using polyclonals from Cell Signaling Technology (part of NEB). The method appears to be very clean and robust, with an approximately 30-fold increase in mean channel number for pERK1/2 following PMA activation of PBL's. I can send you the protocols if you wish. David Hedley Ontario Cancer Institute/Princess Margaret Hospital Toronto. > -----Original Message----- > From: joan Kalnitsky [SMTP:jkalnits@vt.edu] > Sent: Friday, March 09, 2001 2:21 PM > To: Cytometry Mailing List > Subject: MAP-K > > > > I have a client who has been valiantly trying to use the Biosource > Int. > Anti ERK 1/2(pTpY1851187) FITC labeled antibody to measure MAP-K. She is > using freshly isolated human PBMC's. The Biosource booklet shows a nice > overlay of histograms with Jurkat cells, both unstimultated and PMA > stimulated. The PMA stimulated histogram being (what appears to be) much > greater. The booklet states that fresh human PBMC's can also be used in > this manner. She is seeing no difference in her stimulated and > unstimulated samples. When she calls to talk to the company she is not > getting much help. > I am wondering if anyone is using this asay and if you would have > any > suggestions or ideas. > Thanks in advance, > Joan K > > > > Flow Cytometry Lab Supervisor > VMRCVM > (540) 231-4115 > FAX 540-231-7367 > jkalnits@vt.edu > > "It is better to serve than to receive." > B. Borg
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