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PacBlue succinimidyl ester [equivalent to Pacific Blue succinimidyl ester]

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

Protein stock solution (Solution A)
  1. To prepare a protein labeling stock solution, 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., an antibody with a protein concentration >2 mg/mL, if possible). This will give you a 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.

PacBlue succinimidyl ester stock solution (Solution B)
  1. 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 a 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 a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.

Run conjugation reaction
  1. Use a 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 a 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too low or too high, determine the optimal dye/protein ratio at 5:1, 15:1, and 20:1 respectively.

  2. 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.

  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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 needs to be diluted with staining buffer, and aliquoted for multiple uses.

    Note: For longer-term storage, dye-protein conjugate solution needs to 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 mg0.5 mg1 mg5 mg10 mg
1 mM294.803 µL1.474 mL2.948 mL14.74 mL29.48 mL
5 mM58.961 µL294.803 µL589.605 µL2.948 mL5.896 mL
10 mM29.48 µL147.401 µL294.803 µL1.474 mL2.948 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum

Page updated on November 12, 2024

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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-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

CAS

215868-33-0
Flow cytometry analysis of PBMC stained with PacBlue anti-human CD3 *SK7* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the PacBlue specific V3-A channel.
Flow cytometry analysis of PBMC stained with PacBlue anti-human CD3 *SK7* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the PacBlue specific V3-A channel.
Flow cytometry analysis of PBMC stained with PacBlue anti-human CD3 *SK7* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the PacBlue specific V3-A channel.
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