Hi Dr. Roy and fellow flowers: I do this all the time. I have abandoned Western blot in favor of intracellular flow cytometry, and I have compared indirect to direct staining, with similar results. I use either a biotinylated first step or an unlabeled first step. If I use an unlabeled, I use a ligand-affinity-purified polyclonal, and a labeled F(ab2') for the second step. I block with Ig of the same species as the F(ab2'). I have successfully seen p38, STAT3, JAK2, and a lot of cytokines. The first step does not have to be one specifically for flow to work--if it works in westerns and immunohistochemistry chances are excellent that the Ab will work here too. A major pitfall is not blocking sufficiently and not washing sufficiently. I block with several mg/ml Ig for about 1 hr on ice after permeabilization. Also, the first wash after adding Ig, Ab, or second step must be with buffer added and left there for the same length of time as the incubation step. For example, if you left Ab on for 45 min, you must spin out the Ab and then leave the wash buffer on for 45 min, then proceed with the subsequent washes as usual. Another thing I found was that Caltag fix & permeabilization reagents gave me superior signal to noise ratios for phospho-Ab labeling, while Pharmingen or home-made reagents gave me very good results for intracellular cytokines. See these references for more details: Fleisher et al., Clin Immunol 90:425-430, 1999 Barton & Murphy Cytokine 12:18-27, 2000 Feel free to contact me for more info. Best, Beverly Barton Assistant Professor Dept. of Surgery UMDNJ-New Jersey Medical School Meenakshi Roy wrote: > > Dear Flow-ers, > I recently read the paper "The impact of duration versus extent of > TCR occupancy on T cell activation: a revision of the kinetic proofreading > model" by Rosette et al in Immunity Vol 15, July 2001. > Here they describe intracellular staining for phospho-Jun and phospho > MAPK, after T cell activation. > I would like to reproduce this. Has anyone here experience with doing > this? What are the pitfalls? Also, the autohors describe unconjugated > primary antibodies followed by biton secondary and strep-PE. Anyone know > where I can obtain a FITC-conjugated anti-MAPK that would work for this? > The clones they use are: > anti-phospho-p44/p42 MAPK (E10) > anti-phopspho JNK (G-7) > > What other phospho-antibodies work well for this? > Regards, > Dr. Meenakshi Roy > UCLA
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