The Goat anti-mouse IgG isotype specific polyclonal Abs from Caltag are excellent. We have used the anti-IgG1, IgG2a and IgG2b- all with good results [Journal of Immunology, 159, 5862-5870 (1997)]. These mAbs show excellent specificity (e.g. the anti-IgG1 does not recognize an IgG2a). All of these Abs work best as PE, PE/Cy5 or APC conjugates, so unless you absolutely have to, don't use FITC. All of these secondary mAbs MUST be titrated to give you the best S/N- have you done that? Saponin must be present in all wash buffers, however some of the commercial kits use stronger detergents which cause longer lasting permeabilization. I'd be interested in hearing from others about saponin as well. Calman > ---------- > From: Ed Fulton > Sent: Friday, May 21, 1999 19:37 > To: Cytometry Mailing List > Subject: 1)Isotype specific secondary and 2)Saponin > > > Dear FLOWers, > > I've been doing 3 color immunofluorescence on a single laser (488nm) > benchtop analyzer looking at a surface marker, an intracellular antigen, > and BrdU in the DNA of sheep PBMCs. The technique was working fine until > I decided to look at more surface markers. The previous surface and > internal marker antibodies were of different species (goat and mouse) so I > was able to use anti-goat and anti-mouse secondaries. Now however, I've > had to use two mouse antibodies (sheep antibodies aren't all that common) > so I've had to use an IgG2a and an IgG1 isotype specific secondary. The > surface/IgG2a part is working fairly well but the internal/IgG1 isn't > working. Two possible explanations are that my previous internal > secondary > was Mouse IgG(heavy and light chain) which may have more binding sites > than the IgG1 isotype antibody or that the previous antibody was a F'(ab)2 > while the new antibody is whole and may have trouble getting into the > cell. So the first question is: > > 1) Has anyone had any experience (good or bad) using isotype specific > secondary antibodies for surface and intracellular staining that they > could share? > > I've been doing the above staining using commercial fixation and > permeabilization reagents but for money and flexibility reasons I'd like > to prepare my own reagents in the future. So with that in mind, the > second question is: > > 2) Can anyone recommend a source and grade of saponin to be used as > permeabilization for internal antigen staining. > > For example, I've seen that Pharmingen and Sigma sell saponin but some of > their products have differing levels of Sapogenin and low-molecular weight > contaminants. I'm not up on Saponin chemistry so maybe someone could > enlighten me. Also, how important is it to keep saponin around during all > the staining steps? I used the permeabilization reagent only with the > primary internal antibody previously and not with the secondary. However, > I may have been able to get away with that since I was using Fab2 but the > whole secondary ab may need the continued presence of a detergent to allow > entry into the cytoplasm. > > Thanks for any insights and sorry for the long explanation, > > Ed Fulton > Grad. Student > Animal Science > U.C. Davis > befulton@yahoo.com (check more often) > befulton@ucdavis.edu >
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