I agree that I/C staining with biotinylated primary Abs can be less than optimal, when compared to directly labeled Abs. 1. Have you titered the polyclonal? Non-specific binding is very concentration dependent. 2. Is this an affinity purified polyclonal (using a column bound with Ag)? Affinity purified Abs have much lower background staining. The typical problem I have gotten is specific binding of the SA to endogenous cellular biotin. You can test that by adding only the SA-APC (no Ab) and seeing the level of staining. My guess is that will add 1/2 to 1 log, but not 3 logs of noise. To reduce the endogenous biotin, one adds an excess of unlabeled SA, wash x 2 and then add an excess of free biotin. You describe only adding the SA. If you have not subsequently added free biotin this WILL increase noise substantially. SA is tetravalent, so adding it just creates more biotin binding sites that will bind the biotinylated control. Performing the blocking without added biotin could increase your noise 3 logs. I cannot remember the exact conditions, but it should be in a JI paper I published in 1995 (check Pubmed). While we are on the subject, the best control for your specific staining is not a irrelevant goat Ig control. As per the isotype control discussion of the last few weeks, these are imperfect at best. The fact that you are performing I/C staining with polyclonal Abs in a system with dim receptors makes it even worse. The best control is to do a blocking experiment, either with unlabeled specific Ab or with the ligand. If the polyclonal was made to a peptide it is real easy to do the latter. If you need to use recombinant receptor, you may be out of luck. I can send you a detailed protocol if you are interested. Use of only an "isotype match control" to validate the specificity of I/C staining is not sufficient. Blocking controls must be done. Hey Mario, how's that for dogma? Calman > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > > ---------- > From: Wolfraim, Lawrence (NCI) > Sent: Saturday, November 17, 2001 2:42 PM > To: Cytometry Mailing List > Subject: IC flow with biotinylated primary > > > Dear Flowers, > > I am attempting to analyze surface and intracellular levels of the > TGF-beta > type II receptor (RII) by flow and intracellular flow. I am using a > biotinylated primary antibody (a goat polyclonal unfortunately) and then > coming back with a stretavidin comjugate (APC -strep). For surface > staining, I do get a nice shift relative to a "normal" biotinylated goat > polyclonal (my "isotype" control). > > For the intracellular staining, even when I pre-incubate (in > permeabilization buffer, containing saponin) with unconjugated > streptavidin > (to reduce background) I still see a significant shift with my > biotinylated > "control". The MFI is around the 3rd log decade, which is bright. No > further shift is seen with the biotinylated anti-RII. To my knowledge, no > directly conjugated primary antibodies are available against the > extracellular domain of RII. > > Does anyone out there have any experience in: > > (1) looking at surface or IC TGF-beta type II receptor levels by flow > cytometry; &/or > > (2) using biotinylated antibodies in surface staining or IC flow? > > Any help would be greatly appreciated. > > Thanks, > > Lawrence Allen Wolfraim, Ph.D. > Laboratory of Cell Regulation & Carcinogenesis (LCRC) > National Cancer Institute/National Institutes of Health > 41 Library Drive > Building 41, Room B910 > Bethesda MD 20892 > Tel.: (301) 496-2431 > Fax: (301) 496-8395 > wolfrail@mail.nih.gov <mailto:wolfrail@mail.nih.gov> > >
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:40 EST