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iFluor® Ultra 594 maleimide

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Physical properties
Molecular weight1097.12
SolventDMSO
Spectral properties
Absorbance (nm)585
Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.05
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)586
Emission (nm)601
Quantum yield0.911
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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iFluor® 840 goat anti-mouse IgG (H+L)
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iFluor® 860 goat anti-mouse IgG (H+L)
iFluor® 860 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 800 goat anti-rabbit IgG (H+L)
iFluor® 800 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 810 goat anti-rabbit IgG (H+L)
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iFluor® 610 succinimidyl ester
iFluor® 710 succinimidyl ester
iFluor® 790 succinimidyl ester
iFluor® 800 succinimidyl ester
iFluor® 810 succinimidyl ester
iFluor® 820 succinimidyl ester
iFluor® 860 succinimidyl ester
iFluor® 546 succinimidyl ester
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iFluor® 430 succinimidyl ester
iFluor® 450 succinimidyl ester
iFluor® 840 succinimidyl ester
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iFluor® 720 succinimidyl ester
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iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 532-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 680-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 700-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 750-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 790-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 570 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*
iFluor® 670 Styramide *Replacement for Opal 690*
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iFluor® 790 Azide
iFluor® 790 Alkyne
iFluor® 720 maleimide
Show More (281)

OverviewpdfSDSpdfProtocol


Molecular weight
1097.12
Absorbance (nm)
585
Correction Factor (260 nm)
0.07
Correction Factor (280 nm)
0.05
Extinction coefficient (cm -1 M -1)
1800001
Excitation (nm)
586
Emission (nm)
601
Quantum yield
0.911
The iFluor® Ultra series represents an enhancement of our established iFluor® dyes, optimized for antibody labeling in fluorescence imaging and flow cytometry. Within this series, iFluor® Ultra 594 is an exceptionally bright red fluorescent dye excitable by 561 nm or 594 nm laser lines, with a peak emission at 601 nm. Antibody conjugates with iFluor® Ultra 594 exhibit superior brightness compared to those labeled with Alexa Fluor® 594 under identical conditions. Additionally, iFluor® Ultra 594 maintains stable fluorescence across a pH range of 4 to 10. The maleimide derivative of iFluor® Ultra 594 is widely used for conjugation to thiol groups on proteins, oligonucleotide thiophosphates, or low molecular weight ligands. Conjugates with iFluor® Ultra 594 demonstrate higher fluorescence intensity and greater photostability compared to those with other spectrally similar fluorophores, enhancing their utility for advanced fluorescence applications. Fluorescent dye-conjugated antibodies are crucial for protein identification in various applications, including fluorescent cell imaging, flow cytometry, western blotting, and immunohistochemistry. The advantages of these conjugates include increased sensitivity, multiplexing capabilities, and ease of use, facilitating complex biological studies.

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

iFluor® Ultra 594 maleimide Stock Solution (Solution B)
  1. Prepare a 10 mM iFluor® Ultra 594 maleimide stock solution by adding anhydrous DMSO to the vial of iFluor® Ultra 594 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 iFluor® Ultra 594 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 iFluor® Ultra 594 maleimide per mole of antibody. The following steps outline how to determine the DOS of iFluor® Ultra 594 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 = 601 nm for iFluor® Ultra 594 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 iFluor® Ultra 594 maleimide, it is at 601 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

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® Ultra 594 maleimide 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 mM91.148 µL455.739 µL911.477 µL4.557 mL9.115 mL
5 mM18.23 µL91.148 µL182.295 µL911.477 µL1.823 mL
10 mM9.115 µL45.574 µL91.148 µL455.739 µL911.477 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Absorbance (nm)585
Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.05
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)586
Emission (nm)601
Quantum yield0.911

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® Ultra 647 maleimide65567025000010.3910.070.07
iFluor® Ultra 750 maleimide74977325000010.3210.040.05

References


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