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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web