Microsphere Surface Protein Determination Using Flow Cytometry

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 13.2.1 Excitation (solid) and emission (dashed) spectra of ATTO‐TAG CBQCA.

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Figure 13.2.2 CBQCA staining of protein microsphere standards and linear fit.

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Figure 13.2.3 Measurement of specific binding of a solution‐phase protein to a conjugated microsphere set via the use of a fluorescent ligand.

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Figure 13.2.4 Linear fit of a MESF (molecules of equivalent soluble fluorochrome) standard curve using fluorescein isothiocyanate (FITC) standard microspheres.

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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.
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	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.
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	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.