PacBlue succinimidyl ester [equivalent to Pacific Blue succinimidyl ester]

Flow cytometry analysis of PBMC stained with PacBlue anti-human CD3 *SK7* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PacBlue specific V3-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	339.21
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.15
Correction Factor (280 nm)	0.2
Extinction coefficient (cm ^-1 M ^-1)	46000
Excitation (nm)	404
Emission (nm)	455
Quantum yield	0.78

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

CAS

215868-33-0

Molecular weight

339.21

Correction Factor (260 nm)

0.15

Correction Factor (280 nm)

0.2

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

46000

Excitation (nm)

404

Emission (nm)

455

Quantum yield

0.78

PacBlue succinimidyl ester is the same molecule to Pacific Blue™ succinimidyl ester (Pacific Blue™ is the trademark of ThermoFisher). The amine-reactive Pac Blue succinimidyl ester can be used to prepare the blue fluorescent antibody conjugates that can be well excited by the 405 nm spectral line of the blue diode (violet) laser. These antibody conjugates are widely used in flow cytometry applications.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution. Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

2. PacBlue succinimidyl ester stock solution (Solution B)

Add anhydrous DMSO into the vial of PacBlue succinimidyl ester to make a 10 mM stock solution. Mix well by pipetting or vortex. Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with PacBlue succinimidyl ester. You might need further optimization for your particular proteins. Note: Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.

Run conjugation reaction

Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. Note: We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify the conjugation

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate. Note: For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. Note: For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of PacBlue succinimidyl ester [equivalent to Pacific Blue succinimidyl ester] to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	294.803 µL	1.474 mL	2.948 mL	14.74 mL	29.48 mL
5 mM	58.961 µL	294.803 µL	589.605 µL	2.948 mL	5.896 mL
10 mM	29.48 µL	147.401 µL	294.803 µL	1.474 mL	2.948 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Correction Factor (260 nm)	0.15
Correction Factor (280 nm)	0.2
Extinction coefficient (cm ^-1 M ^-1)	46000
Excitation (nm)	404
Emission (nm)	455
Quantum yield	0.78

Images

Figure 1. Flow cytometry analysis of PBMC stained with PacBlue anti-human CD3 *SK7* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PacBlue specific V3-A channel.