Direct fluorescent staining and analysis of proteins on microspheres using CBQCA.
BACKGROUND
General methods for accurate determination of microsphere surface protein loading are needed for applications from protein arrays to molecular assembly studies. Current methods include bulk absorption measurements of stained microspheres or use of known fluorescently tagged binding partners, which limit sensitivity and general applicability, respectively.


METHODS
Microspheres bearing covalently coupled proteins were stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) using different incubation times and dye concentrations to determine optimal staining conditions. The CBQCA fluorescence of microspheres (measured by flow cytometry) bearing known amounts of protein were used to generate standard curves of CBQCA fluorescence response versus the amount of microsphere surface protein. CBQCA was also used to stain noncovalent protein interactions.


RESULTS
Maximal labeling was attained within 1 h with 1 mM CBQCA. Linear fluorescence response occurred between 8 x 10(4) and 1 x 10(6) proteins/microsphere. CBQCA staining did not disturb noncovalent protein interactions.


CONCLUSIONS
We have developed methods using CBQCA and flow cytometry to quickly and simply quantify the amount of protein on the surface of a microsphere. Importantly, this approach could be extended to other formats (e.g., chips). Further, because it does not disturb noncovalent protein-protein interactions, it may be possible to use this approach to detect protein interactions without the use of purified prestained probes.
Graves
Steven W
SW
National Flow Cytometry Resource, Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico. graves@lanl.gov
Woods
Travis A
TA
Kim
Heungbok
H
Nolan
John P
JP
D000818:Animals; D001360:Avidin / Q000737:chemistry; D001565:Benzoates / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D005434:Flow Cytometry / Q000379:methods; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008863:Microspheres; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013499:Surface Properties; D013997:Time Factors
D001565:Benzoates; D005456:Fluorescent Dyes; C029152:G-substrate; D007074:Immunoglobulin G; D008024:Ligands; D009419:Nerve Tissue Proteins; D011506:Proteins; D011804:Quinolines; C069617:3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; D001360:Avidin; D001710:Biotin