ReadiUse™ Preactivated PE-Texas Red Maleimide

Flow cytometry analysis of whole blood stained with PE-Texas Red anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE-Texas Red specific B6-A channel.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	N/A
Solvent	Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	613

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure

Overview SDS Protocol

Molecular weight

N/A

Extinction coefficient (cm ^-1 M ^-1)

1960000

Excitation (nm)

565

Emission (nm)

613

PE-Texas Red is a popular tandem color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at 600 nm. AAT Bioquest offers this preactivated PE-Texas Red maleimide to facilitate the PE-Texas Red tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. It is prepared from the commonly used crosslinker SMCC. Our preactivated PE-Texas Red maleimide is ready to conjugate the reduced antibodies and other thiol-containing biomolecules.

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Reduction of IgG

Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody solutions should be at 2 mg/mL or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 2 mg/mL.
Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA. (Desalting column: https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500)
Measure the Antibody concentration with Nanodrop. (Con. (mg/ml)= A280nm/1.4) Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification.

Conjugate with ReadiUse™ Preactivated PE-Texas Red Maleimide

Reconstitute ReadiUse™ Preactivated PE-Texas Red Maleimide in 100 µL ddH₂O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated PE-Texas Red Maleimide are stable at 4 °C for one week, please kept it from light.
Add reduced antibody to pre-activated PE-Texas Red directly to at the ratio of 250-300 µg PE-Texas Red /100 µg reduced antibody.
Rotate the mixture for 60-120 mins at room temperature.
After 60 minutes, block the free sulfhydryls on the antibody.
Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature.

Purification

Antibody/PE-Texas Red conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/PE-Texas Red conjugate solution is recommended to be stored at 2~8 °C and kept from light.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	613

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated PE-Texas Red Tandem	565	613	1960000

Images

Figure 1. Flow cytometry analysis of whole blood stained with PE-Texas Red anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE-Texas Red specific B6-A channel.

References

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Journal: Cytometry (1991): 570-8

Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry.
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Journal: Cytometry (1991): 350-9