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G. Durack
UIUC Biotechnology Center
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I am interested in quantitating the number of functional proteins
covalently bound to the surface of polystyrene beads using fluorescence
flow cytometry. In particular, I am interested in comparing the relative
amounts of protein between the two bead populations. I have 2
protein-coated bead populations: beads coated with Protein A and beads
coated with Human IgG. In order to measure the number of sites on the
Protein A-coated beads, I have probed with a FITC-Human IgG. (The assay
involves incubating the Protein-A coated beads over a range of FITC-Human
IgG concentrations, and then calculating Rtotal (the 'total' number of
proteins, in MESF units) and Kd. In order to measure the number of sites
on the Human IgG -coated beads, I have probed with a FITC-Protein A. My
goal is now to convert the Rtotal's from MESF units to actual protein
number. At a minimum, I need to be able to compare the ratio of
MESF/protein A to MESF/Human IgG. Clearly, the manufacturer f/p ratio is
not sufficient. I am seeking a technique to convert the MESF values to
actual protein number using flow cytometry.
Any suggestions would be greatly appreciated.
saterbak@aries.scs.uiuc.edu
217-244-2007
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Gary Durack
University of Illinois Biotechnology Center
Voice (217) 244-0559
FAX (217) 244-0466
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