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Hi, The blocking conditions was 5% fat free milk in a solution of 50mM TRIS pH7.5, tween 20 (0.05%) and NaCl (150Mm), at room temperature for 1 hour. The cystathionase antibody concentration I used initially was 2ug/ml, however, I saw no bands, and so then increased this to 5ug/ml. Still no bands were seen. The anti mouse secondary antibody was purchased from sigma cat no: A9044 and diluted 1:4500. The diluent was the same as the blocking solution (freshly prepared). Incubation was for 1.5 hours at room temperature. And yes, this blot is showing CBS at 63Kda. (This CBS antibody was produced in mouse, and was detected by our anti mouse secondary ab from sigma). I first tried detecting Cystathionine on this blot and saw no bands. I then re-carried out w.blotting using fresh lysates, and used the higher anitbody concertration, but was still unable to detect the cystathionase.
Asked on Oct 13 2011
Thank you for sending these additional details. If we assume the cystathionase in your samples is intact and not degraded then the antibody is probably the issue. I think your protocol is correct, and the Hela lysate, at least, should be giving a signal. Would you be interested in trying the rabbit polyclonal, ab80643, as a free-of-charge replacement? The only data we have is a blot against transfected 293 cells, but we will offer our guarantee (replacement or credit or refund within 6 months of purchase, after considering the protocol) for reactivity with endogenous cystathionase in human samples, e.g. HeLa. Reactivity with other species is predicted based on homology with the immunogen but has not been tested.
Answered on Oct 13 2011