Thermo Fisher Scientific R-Phycoerythrin User guide

Brand: Thermo Fisher Scientific

Model: R-Phycoerythrin

Type: User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

46185 R-Phycoerythrin, 2mg supplied as an ammonium sulfate suspension

Molecular Weight: 240K

Extinction Coefficient: 2.0 x 106 M-1cm-1

Storage: Upon receipt store at 4°C protected from light. Do not freeze. Product shipped with an ice

pack.

Introduction

Thermo Scientific R-Phycoerythrin, a red fluorescent, multi-subunit protein, is used for fluorescent conjugation. This

fluorochrome is a member of the phycobiliprotein family of proteins isolated from the marine algae Porphyra tenera or

Gastroclonium coulterii. Fluorochrome-labeled reagents have high resolution and are therefore advantageous in

immunological assays, such as live cell staining, double-labeling techniques and cell sorting procedures. The strong

absorption bands of the R-Phycoerythrin are in the visible region of the spectrum, extending from the green to the far-red

wavelengths. The absorbance spectra extends over a broad range of potential excitation wavelengths, allowing for versatility

in the excitation source and creating large Stokes shifts, thus minimizing interference from Rayleigh-scattered light.1,2

R-Phycoerythrin conjugates can be used with three- and four-color fluorescence activated cell sorting.3-6 R-Phycoerythrin is

excited with the argon ion laser at 488nm and detected at 570nm. The excitation and emission spectrum of R-Phycoerythrin

is shown in Figure 1.

The fluorescence of R-Phycoerythrin is not quenched by naturally occurring biological components. The protein is soluble in

aqueous environments and exhibits minimal nonspecific binding. R-Phycoerythrin has 34 bilin groups which gives the

reagent a high absorbance coefficient. A high fluorescence quantum yield of 0.82 at 578nm is independent of temperature

and pH over a broad range. The spatial arrangement of (αΒ)6γ of the bilin structure within the protein prevents concentration

quenching and provides steric protection that enables the fluorescence to be independent of pH.1,7

Figure 1. The excitation and emission spectra of R-Phycoerythrin.

0350.2

46185

R-Phycoerythrin

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Example Protocol for Preparing Pyridyldisulfide Activated Phycobiliproteins

Note: This protocol will require optimization for specific applications. A variety of cross-linkers can be used to prepare

phycobiliprotein conjugates.4 The method below uses SPDP, LC-SPDP or Sulfo-LC-SPDP. These cross-linkers react with

primary amines on the surface of the phycobiliprotein and introduce pyridyldisulfide group(s) that can be reacted with

another protein that contains a free sulfhydryl.

Materials Required

• Desalting columns, such as Thermo Scientific Dextran Desalting Columns (Product No. 43230)

• Dialysis cassette: (Thermo Scientific Slide-A-Lyzer Dialysis Cassettes, Product No. 66382, 66372 or 66332)

• Cross-linker such as SPDP (Product No. 21857), LC-SPDP (Product No. 21651) or Sulfo-LC-SPDP (Product No. 21650)

• Phosphate buffered saline (PBS): 0.1M sodium phosphate, 150mM NaCl at pH 7.2 - 7.5 (e.g., Thermo Scientific BupH

Phosphate Buffered Saline Packs, Product no. 28372)

• SPDP, LC-SPDP or Sulfo-LC-SPDP solution: 20mM SPDP or LC-SPDP in DMSO or 10mM Sulfo-LC-SPDP in water.

Sulfo-LC-SPDP is water-soluble and can be added directly to the phycobiliprotein solution

• Borate buffered saline (BBS): 50mM sodium borate, 300mM NaCl, pH 8.5

• Phycobiliprotein solution: Dialyze R-Phycoerythrin into BBS and adjust protein concentration to 1mg/mL. Note: Protein

is supplied in an ammonium sulfate suspension; extensive dialysis is necessary to remove extraneous ammonium ion,

which will quench the cross-linking reaction.

Procedure

1. Add 25µL of the 20mM SPDP solution to 1mg of phycobiliprotein in 1.0mL BBS.

Note: 0.25-0.30mg of the water-soluble cross-linker Sulfo-LC-SPDP can be added directly to the phycobiliprotein

solution.

2. React for 30 minutes at room temperature.

3. Remove non-reacted cross-linker by applying the sample to a 5mL desalting column equilibrated with PBS. Add PBS

and collect 1mL fractions. The first peak to emerge from the column, as measured by the absorbance at 280nm, will be

the pyridyldisulfide-activated phycobiliprotein.

4. The pyridyldisulfide-activated phycobiliprotein can be reacted with another protein containing a free sulfhydryl using the

protocol for the specific cross-linker used.

Cited References

1. Glazer, A. N. and Stryer, L. (1984). Phycofluor probes. Trends Biochem Sci 9:423-7.

2. Kronick, M. N. and Grossman, P. D. (1983). Immunoassay techniques with fluorescent phycobiliprotein conjugates. Clin Chem 29:1582-6.

3. Lanier, L. L. and Loken, M. R.(1984). Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry

analysis: Correlation of Leu-2, Leu-3, Leu-7, and Leu-11 cell surface antigen expression. J Immunol, 132:151-6.

4. Parks, D. R., et al. (1984). Three-color immunofluorescence analysis of mouse B-lymphocyte subpopulations. Cytometry 5:159-68.

5. Hardy, R. R., et al. (1983). Demonstration of B-cell maturation in X-linked immunodeficient mice by simultaneous three-color immunofluorescence.

Nature 306:270-2.

6. Hardy, R. R., et al. (1984). J Exp Med 159:1169-88.

7. Kronick, M. N. (1986). The use of phycobiliproteins as fluorescent labels in immunoassay. J Immuno Meth 92:1-13.

General References

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. p. 409.

Oi, V. T., et al. (1982). Fluorescent phycobiliprotein conjugates for analysis of cells and molecules. J Cell Biol 93:981-6.

Yeh, S. W., et al. (1987). Fluorescence properties of allophycocyanins and a crosslinked allophycocyanin trimer. Cytometry 8:91.

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

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as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and

workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications

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