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ATTO 488 maleimide

ATTO 488 is a hydrophilic, rhodamine-based fluorescent dye with exceptional water solubility. It is characterized by strong absorption, a high fluorescence quantum yield, and exceptional photostability, making it highly suitable for advanced fluorescence imaging techniques. The dye exhibits optimal excitation within the 480-515 nm wavelength range, aligning precisely with the 488 nm emission line of the Argon-Ion laser. ATTO 488 is particularly effective for single-molecule detection and super-resolution microscopy methods such as PALM, dSTORM, and STED. Moreover, it is well-suited for flow cytometry (FACS), fluorescence in situ hybridization (FISH), and other bioanalytical applications.

The maleimide derivative of ATTO 488 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

ATTO 488 maleimide Stock Solution (Solution B)
  1. Prepare a 10 mM ATTO 488 maleimide stock solution by adding anhydrous DMSO to the vial of ATTO 488 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 ATTO 488 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 ATTO 488 maleimide per mole of antibody. The following steps outline how to determine the DOS of ATTO 488 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 = 520 nm for ATTO 488 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 ATTO 488 maleimide, it is at 520 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)
iFluor® 488 maleimide4915167500010.910.210.11
ATTO 532 maleimide5315521150000.900.220.11
ATTO 647 maleimide6466661200000.200.080.04
ATTO 647N maleimide6456631500000.6510.060.05
ATTO 594 maleimide6026211200000.850.260.51
ATTO 514 maleimide510531115,0000.850.210.08
ATTO 565 maleimide5625891200000.900.270.12
ATTO 488 DBCO499520900000.800.220.09
ATTO 390 maleimide39047524000.900.460.09
ATTO 425 maleimide438484450000.900.190.17
ATTO 495 maleimide497525800000.20.450.37
ATTO 550 maleimide5535741200000.800.230.10
ATTO 590 maleimide5926211200000.800.390.43
ATTO 610 maleimide6156321500000.700.030.06
ATTO 620 maleimide61964112000010.510.040.06
ATTO 633 maleimide6296511300000.6410.040.05
ATTO 655 maleimide6616791250000.310.240.08
ATTO 488 Tetrazine499520900000.800.220.09
ATTO 488 TCO499520900000.800.220.09
ATTO 680 maleimide6796961250000.300.300.17
ATTO 700 maleimide6997151200000.250.260.41
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Page updated on October 28, 2024

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Physical properties

Molecular weight

825.74

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.22

Correction Factor (280 nm)

0.09

Extinction coefficient (cm -1 M -1)

90000

Excitation (nm)

499

Emission (nm)

520

Quantum yield

0.80

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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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.