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AAT Bioquest

XFD532 C5 Maleimide *Same Structure to Alexa Fluor™ 532 C5 Maleimide*

XFD532, manufactured by AAT Bioquest, is structurally identical to Alexa Fluor™ 532 (ThermoFisher). It is a bright yellow-fluorescent dye with an excitation optimized for use with the 532 nm line of the frequency-doubled Nd:YAG laser. XFD532 demonstrates good aqueous solubility and pH-insensitivity over a broad pH range (pH 4–10), ensuring stable fluorescence generation under varying experimental conditions. This dye is particularly suited for multicolor fluorescence microscopy and flow cytometry, as well as advanced applications in super-resolution imaging techniques such as dSTORM.

The maleimide derivative of XFD532 is widely used for labeling biomolecules with free thiol (SH) groups, including antibodies, proteins, thiol-modified oligonucleotides, and low molecular weight ligands. Maleimides react readily with sulfhydryl groups, forming stable thio-ether bonds between the dye and the biomolecule, facilitating robust and reliable labeling for diverse experimental 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

XFD532 C5 maleimide Stock Solution (Solution B)
  1. Prepare a 10 mM XFD532 C5 maleimide stock solution by adding anhydrous DMSO to the vial of XFD532 C5 maleimide. Mix well by pipetting or vortexing.

    Note: Before starting the conjugation process, prepare the dye stock solution (Solution B) and use it promptly. Prolonged storage of Solution B may reduce its activity. If necessary, Solution B can be stored in the freezer for up to 4 weeks, provided it is protected from light and moisture. Avoid freeze/thaw cycles.

Protein Stock Solution (Solution A)
  1. Prepare a 1 mL protein labeling stock solution, by mixing 100 µL of a reaction buffer (e.g., 100 mM MES buffer with a pH ~6.0) with 900 µL of the target protein solution (e.g., an antibody or protein solution with a concentration >2 mg/mL if possible).

    Note: The pH of the protein solution (Solution A) should be 6.5 ± 0.5.

    Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.

    Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. To achieve optimal labeling efficiency, it is recommended to maintain a final protein concentration within the range of 2-10 mg/mL.

Disulfide Reduction (If Necessary)

If your protein does not contain a free cysteine, it must be treated with DTT or TCEP to generate a thiol group. DTT and TCEP are utilized to convert disulfide bonds into two free thiol groups. If using DTT, ensure to remove any free DTT via dialysis or gel filtration before conjugating a dye maleimide to your protein. Below is a sample protocol for generating a free thiol group:

  1. To prepare a fresh solution of 1 M DTT, dissolve 15.4 mg of DTT in 100 µL of distilled water.

  2. To prepare the IgG solution in 20 mM DTT, first, add 20 µL of DTT stock to each milliliter of the IgG solution while mixing gently. Then, allow the solution to stand at room temperature for 30 minutes without additional mixing. This resting period helps to minimize the reoxidation of cysteines to cystines.

  3. Pass the reduced IgG through a filtration column that has been pre-equilibrated with "Exchange Buffer." Collect 0.25 mL fractions as they elute from the column.

  4. Determine the protein concentrations and combine the fractions containing the highest amounts of IgG. This can be accomplished using either spectrophotometric or colorimetric methods.

  5. Proceed with the conjugation immediately after this step (refer to the Sample Experiment Protocol for details).

    Note: IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column.

    Note: The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate, or TRIS buffers.

    Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was designed for the conjugation of goat anti-mouse IgG with XFD532 C5 maleimide. You may need to further optimize the protocol for your specific proteins.

Note: Each protein requires a specific dye-to-protein ratio, which varies based on the properties of the dyes. Over-labeling a protein can negatively impact its binding affinity while using a low dye-to-protein ratio can result in reduced sensitivity.

Run the 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) to the vial of the protein solution (95 µL of Solution A), and mix thoroughly by shaking. The protein solution has a concentration of ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.

    Note: We recommend using a 10:1 molar ratio of Solution B (dye) to Solution A (protein). If this ratio is not suitable, determine the optimal dye/protein ratio by testing 5:1, 15:1, and 20:1 ratios.

  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify the Conjugate

The following protocol serves as an example for purifying dye-protein conjugates using a Sephadex G-25 column.

  1. Follow the manufacturer's instructions to prepare the Sephadex G-25 Column.

  2. Load the reaction mixture (from the "Run conjugation reaction" step) onto 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 of the resin surface.

  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Then, combine the fractions that contain the desired dye-protein conjugate.

    Note: For immediate use, dilute the dye-protein conjugate with staining buffer. If you need to use it multiple times, divide it into aliquots.

    Note: For long-term storage, the dye-protein conjugate solution should be either concentrated or freeze-dried.

Characterize the Desired Dye-Protein Conjugate

The Degree of Substitution (DOS) is a key factor in characterizing dye-labeled proteins. Proteins with a lower DOS generally have weaker fluorescence intensity, while those with a higher DOS may also have reduced fluorescence. For most antibodies, the optimal DOS is recommended to be between 2 and 10, depending on the properties of the dye and protein. For effective labeling, the DOS should be controlled to have 5-8 moles of XFD532 C5 maleimide per mole of antibody. The following steps outline how to determine the DOS of XFD532 C5 maleimide-labeled proteins.

Measure Absorption

To measure the absorption spectrum of a dye-protein conjugate, maintain the sample concentration between 1 and 10 µM. The exact concentration within this range will depend on the dye's extinction coefficient.

Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 534 nm for XFD532 dyes)

For most spectrophotometers, dilute the sample (from the column fractions) with de-ionized water until the OD values fall within the range of 0.1 to 0.9. The optimal absorbance for protein is at 280 nm, while for XFD532 C5 maleimide, it is at 534 nm. To ensure accurate readings, make sure the conjugate is free of any non-conjugated dye.

Calculate DOS

You can calculate DOS using our tool by following this link:

https://www.aatbio.com/tools/degree-of-labeling-calculator

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
XFD488 C5 Maleimide *Same Structure to Alexa Fluor™ 488 C5 Maleimide*499520710000.9210.300.11
XFD350 C5 Maleimide *Same Structure to Alexa Fluor™ 350 C5 Maleimide*34344119000-0.250.19
XFD594 C5 Maleimide *Same Structure to Alexa Fluor™ 594 C5 Maleimide*590618900000.6610.430.56
XFD405 C5 maleimide40142135,000-0.230.70
XFD430 C5 maleimide43254015,000--0.28
XFD546 C5 maleimide5615721120000.7910.210.12
XFD568 C5 maleimide579603913000.6910.450.46
EDANS C5 maleimide3364555900--0.107

References

View all 39 references: Citation Explorer
Development of new hCaM-Alexa Fluor(R) biosensors for a wide range of ligands
Authors: Velazquez-Lopez, I.; Leon-Cruz, E.; Pardo, J. P.; Sosa-Peinado, A.; Gonzalez-Andrade, M.
Journal: Anal Biochem (2017): 13-22
Synthetic Protocol for AFCS: A Biologically Active Fluorescent Castasterone Analog Conjugated to an Alexa Fluor 647 Dye
Authors: Winne, J. M.; Irani, N. G.; Van den Begin, J.; Madder, A.
Journal: Methods Mol Biol (2017): 21-Sep
Alteration of AMPA Receptor-Mediated Synaptic Transmission by Alexa Fluor 488 and 594 in Cerebellar Stellate Cells
Authors: Maroteaux, M.; Liu, S. J.
Journal: eNeuro (2016)
Alexa fluor-labeled fluorescent cellulose nanocrystals for bioimaging solid cellulose in spatially structured microenvironments
Authors: Grate, J. W.; Mo, K. F.; Shin, Y.; Vasdekis, A.; Warner, M. G.; Kelly, R. T.; Orr, G.; Hu, D.; Dehoff, K. J.; Brockman, F. J.; Wilkins, M. J.
Journal: Bioconjug Chem (2015): 593-601
In vivo visualization of GL261-luc2 mouse glioma cells by use of Alexa Fluor-labeled TRP-2 antibodies
Authors: Fenton, K. E.; Martirosyan, N. L.; Abdelwahab, M. G.; Coons, S. W.; Preul, M. C.; Scheck, A. C.
Journal: Neurosurg Focus (2014): E12
Page updated on October 12, 2024

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Physical properties

Molecular weight

790.90

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.24

Correction Factor (280 nm)

0.09

Extinction coefficient (cm -1 M -1)

81000

Excitation (nm)

534

Emission (nm)

553

Quantum yield

0.611

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
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide, or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide, or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide, or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
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