Updated: January 27, 2007

Dear Colleague,

I regret that our stocks of anti-sera are not large enough that we may share them with other labs.

In our experience, the detection of PERK phosphorylation (or IRE1 phosphorylation) as a marker of ER stress is fraught with great difficulties. In most circumstances the anti-phosphoPERK antisera we have tested are unable to detect the protein in straight immunoblots (detection in acutely-stressed AR42J cells may be an exception to this rule). Furthermore, in most circumstances we have been unable to detect the phosphorylation of PERK or IRE1 in response to the over-expression of an ER client protein. We suspect that the reason for this is the slowish rate at which ER stress ensues (compared to the more rapid induction of ER stress in cells treated with toxins that massively and synchronously perturb ER function). Therefore, to detect PERK activation we are forced to resort to the laborious procedure of immunoprecipitation of PERK from detergent lysates followed by immunoblot. This procedure is difficult and consumes large amount of sample and antiserum. That said, Rockland has recently come out with a rabbit polyclonal serum directed to mammalian PERK, which is reported to work in the aforementioned IP/IB procedure (see Figure 1 in their data sheet). Cell Signaling Technology has recently come out with a monoclonal antibody (#3179) that detects phosph-PERK by direct immunoblot of lysates of stressed cells (they used AR42J cells in the example posted on the web). It is our opinion that in many cases use of downstream markers for ER stress (CHOP, BiP and ATF4) is a better course of action. One efficient way to detect ER stress signaling is to monitor XBP-1 splicing in an RT-PCR method (click here for discussion). On occasion there will be a specific question related to PERK activation that can not be answered by the surrogate markers, however in most cases detecting PERK activation adds little and consumes a lot of time and resources.

Affinity Bioreagents has recently commercialized the 9C8 anti-CHOP MoAb that we had originally developed. They distribute IgG derived from ascites produced with this hybridoma. We have tested their product and found it most suitable for immunoblots. While we have not tested their product for IP, our own preparations of this MoAb have worked well in IP and ICC in the past.

Shenolikar and colleagues had published a paper (Brush et al, Mol Cell Biol. 2003 February; 23(4): 1292–1303) in which they report on the successful application of the SCBT anti-GADD34 H-193 serum to stain endogenous GADD34 in NIH 3T3 cells. This is a feat we have been unable to accomplish using our antiserum that had been raised against the mouse protein.

The Santa Cruz rabbit polyclonal antiserum to XBP-1 (sc-7160) is good too, it detects both the human and the mouse. (Please note that our paper on XBP-1 (Calfon, et al., 2002, Nature; 415:92) contains an unfortunate error in that we used the rabbit polyclonal to XBP-1 (SC-7160) for our successful blots and not the murine monoclonal to XBP-1 (SC-8015) as stated erroneously in the paper. We were unable to detect the endogenous XBP-1 with the SC-8015 monoclonal antiserum).

We no longer carry the anti-TLS reagents, however Dick Goodman's lab from Oregon has agreed to distribute the MoAb we raised to TLS, 4H11. Please contact them at the address below:
Jennifer Butler <butlerje@ohsu.edu>
OHSU Vollum Institute, L-474
3181 S.W. Sam Jackson Park Rd.
Portland, OR 97201-3098
Phone: 503-494-4676

Hope you find this information useful.

David Ron