Are you getting a bimodal pattern (positives and negatives) or just positives? If it is the later, it suggests that your staining may be due to non-specific binding of the polyclonal antiserum to the cells. Such binding is not saturable and would thus exhibit the behavior you have noted. Approaches: How does your pre-immune serum stain? Have you tried a negative control cell line? We sometimes make a 1:1 mix of positive and negative control cells to generate a bimodal pattern which is useful for such titrations. Was the polyclonal Ab was raised against a peptide? If so you can demonstrate specificity by showing that its binding is competed out by a molar excess of peptide. -Happy new year all. Surfs up, gotta' go. Calman > ---------- > From: Art Roberts > Sent: Wednesday, December 29, 1999 5:17 PM > To: Cytometry Mailing List > Subject: Non-saturating polyclonal antibody? > > > Dear Flow-ers, > > I am using a polyclonal antibody (unpurified immune serum) for indirect > staining which gives me a very bright signal on flow. The problem is that > if I stain using serial dilutions of the serum (1:3, 1:10, etc.) the > signal begins to decrease with the first dilution, gradually tapers off, > and reaches the level of the negative control at about 1:10,000. Because > the signal drops with the first dilution, I assume I have not achieved > saturation with the antibody, even though I would expect there to be a > high antibody concentration in the serum. Does anyone have any idea > what's going on? Thanks in advance for any help. > > Art > > __________________________________ > Arthur Roberts > Dept. of Medicine > Robert Wood Johnson Medical School > > email: robertar@umdnj.edu > phone: 732-235-7790 > Fax: 732-235-7792 > > > > -- End -- >
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