Microsphere Surface Protein Determination Using Flow Cytometry

Travis A. Woods1, Steven W. Graves1, John P. Nolan2

1 Los Alamos National Laboratory, Los Alamos, New Mexico, 2 La Jolla Bioengineering Institute, La Jolla, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.2
DOI:  10.1002/0471142956.cy1302s34
Online Posting Date:  November, 2005
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Abstract

This unit describes an extrinsic staining protocol using the amine‐reactive CBQCA dye to measure the amount of protein on the surface of a microsphere. This approach is novel in that it allows microspheres bearing proteins without known binding partners to be accurately quantified on a flow cytometer.

Keywords: flow cytometry; surface chemistry; protein; microspheres

     
 
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Table of Contents

  • Basic Protocol 1: Microsphere Surface Protein Determination
  • Support Protocol 1: Preparation of Protein Microsphere Standards by Coupling at pH 5.5
  • Alternate Protocol 1: Preparation of Protein Microsphere Standards by Coupling with Activation at pH 6.5
  • Support Protocol 2: Characterization of Protein Microsphere Standards
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microsphere Surface Protein Determination

  Materials
  • Calibration microspheres with known amounts of surface‐coupled proteins (Spherotech; or see protocol 2, protocol 4, and protocol 3)
  • Microsphere samples suspended in PBS with 0.05% Tween 20 (see recipe for PBS/0.05% Tween 20)
  • Reaction buffer: 0.1 M sodium borate, pH 9.3, with 0.1% Triton X‐100 (see recipe)
  • 20 mM potassium cyanide (KCN; see recipe)
  • 40 mM ATTO‐TAG CBQCA (see recipe)
  • 1.5‐ml microcentrifuge tubes (preferably siliconized)
  • Rotator, rocker, or other mixing device
  • Flow cytometer analysis tubes
  • Flow cytometer with ∼465‐nm excitation and filters for collection of emission at ∼550 nm

Support Protocol 1: Preparation of Protein Microsphere Standards by Coupling at pH 5.5

  Materials
  • Microspheres, carboxyl‐functionalized polystyrene (Spherotech)
  • Incubation buffer: 0.05 M 2‐(N‐morpholino)ethanesulfonic acid pH 5.5 (MES; Sigma‐Aldrich)
  • N‐hydroxysulfosuccinimide (Sulfo‐NHS; Pierce)
  • 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDAC or EDC; Pierce)
  • IgG (Sigma‐Aldrich)
  • Quench buffer: 0.05 M Tris⋅Cl with 0.05% (v/v) Tween 20
  • Storage buffer: PBS with 0.05% (v/v) Tween 20 (see recipe)
  • 1.5‐ml microcentrifuge tubes (preferably siliconized)
  • Microcentrifuge
  • Rotator, rocker, or other mixing device

Alternate Protocol 1: Preparation of Protein Microsphere Standards by Coupling with Activation at pH 6.5

  • Activation buffer: 0.1 M sodium phosphate buffer, pH 6.3

Support Protocol 2: Characterization of Protein Microsphere Standards

  Materials
  • Protein G (Sigma‐Aldrich)
  • Alexa Fluor 488 microscale protein labeling kit (or a kit with an alternative fluorophore; Molecular Probes)
  • Phophate‐buffered saline (PBS; appendix 2A)
  • Fluorescence standard: fluorescein
  • PBS containing 0.05% Tween 20 (see recipe)
  • IgG‐coupled polystyrene microspheres (Spherotech; or see protocol 2)
  • MESF fluorescence intensity calibration standard microspheres (Bangs Laboratories; http://www.bangslabs.com)
  • Fluorometer cuvettes
  • Fluorometer
  • Flow cytometer analysis tubes (e.g., 12 × 75–mm tubes for a FACScalibur)
  • Flow cytometer with 488‐nm excitation and filters for collection of green emission at 530 nm
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Figures

Videos

Literature Cited

Literature Cited
   Buranda, T., Jones, G.M., Nolan, J.P., Keij, J., Lopez, G.P., and Sklar, L.A. 1999. Ligand receptor dynamics at streptavidin‐coated particle surfaces: A flow cytometric and spectrofluorimetric study. J. Phys. Chem. 103:3399‐3410.
   Graves, S.W., Woods, T.A., Kim, H., and Nolan, J.P. 2005. Direct fluorescent staining and analysis of proteins on microspheres using CBQCA. Cytometry 65A:50‐58.
   Hoffman, R.A. 2001. Standardization and quantitation in flow cytometry. Methods Cell. Biol. 63:299‐340.
   Nolan, J.P. and Sklar, L.A. 2002. Suspension array technology: Evolution of the flat‐array paradigm. Trends Biotechnol. 20:9‐12.
   Sapan, C.V., Lundblad, R.L., and Price, N.C. 1999. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29:99‐108.
   Sklar, L.A., Edwards, B.S., Graves, S.W., Nolan, J.P., and Prossnitz, E.R. 2002. Flow cytometric analysis of ligand‐receptor interactions and molecular assemblies. Annu. Rev. Biophys. Biomol. Struct. 31:97‐119.
   Slomkowski, S. and Basinska, T. 1992. Detection and concentration measurements of proteins adsorbed onto polystyrene and poly(styrene acrolein) latexes. ACS Symposium Series 492:328‐346.
   Templin, M.F., Stoll, D., Schrenk, M., Traub, P.C., Vohringer, C.F., and Joos, T.O. 2002. Protein microarray technology. Trends Biotechnol. 20:160‐166.
   You, W.W., Haugland, R.P., Ryan, D.K., and Haugland, R.P. 1997. 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde, a reagent with broad dynamic range for the assay of proteins and lipoproteins in solution. Anal. Biochem. 244:277‐282.
   Wang, L., Gaigalas, A.K., Abbasi, F., Marti, G.E., Vogt, R.F. and Schwartz, A. 2002. Quantitating fluorescence intensity from fluorophores: Practical use of MESF values. J. Res. Nat. Inst. Stand. Technol. 107:339‐353.
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