Fluorescein-5-maleimide CAS 75350-46-8

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Physical properties

Molecular weight	427.36
Solvent	DMSO

Spectral properties

Absorbance (nm)	487
Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	498
Emission (nm)	517
Quantum yield	0.7900¹, 0.95²

Storage, safety and handling

Certificate of Origin	Download PDF
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

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. Fluorescein-5-maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of Fluorescein-5-maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.

2. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 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. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

3. Optional

if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:

Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol). 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 developed for the conjugate of Goat anti-mouse IgG with Fluorescein-5-maleimide. 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 Fluorescein-5-maleimide *CAS 75350-46-8* 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	233.995 µL	1.17 mL	2.34 mL	11.7 mL	23.399 mL
5 mM	46.799 µL	233.995 µL	467.99 µL	2.34 mL	4.68 mL
10 mM	23.399 µL	116.997 µL	233.995 µL	1.17 mL	2.34 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

Absorbance (nm)	487
Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	498
Emission (nm)	517
Quantum yield	0.7900¹, 0.95²

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)	Correction Factor (482 nm)	Correction Factor (565 nm)
Cyanine 5 maleimide [equivalent to Cy5® maleimide]	651	670	250000¹	0.27¹, 0.4²	0.02	0.03	0.009	0.09
5-TAMRA Maleimide [Tetramethylrhodamine-5-maleimide] CAS 154480-30-5	552	578	90000	-	0.32	0.178	-	-

Images

Figure 1. Chemical structure for Fluorescein-5-maleimide *CAS 75350-46-8*

Citations

View all 4 citations: Citation Explorer

Multifunctional superparamagnetic nanoparticles conjugated with fluorescein-labeled designed ankyrin repeat protein as an efficient HER2-targeted probe in breast cancer
Authors: Li, Dong-Li and Tan, Jian-Er and Tian, Ying and Huang, Shun and Sun, Peng-Hui and Wang, Meng and Han, Yan-Jiang and Li, Hong-Sheng and Wu, Hu-Bing and Zhang, Xing-Mei and others, undefined
Journal: Biomaterials (2017)

Etablierung von zellbasierten Assays zur Identifizierung von Inhibitoren des Chemokins CXCL8
Authors: J{\"o}st, Marina
Journal: (2016)

Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide with Environment Sensitive Drug Release for Effective Treatment of Triple Negative Breast Cancer
Authors: Wu, Xiaohui and Han, Zheng and Schur, Rebecca M and Lu, Zheng-Rong
Journal: ACS Biomaterials Science & Engineering (2016): 501--507

Untersuchungsmethoden f{\"u}r die chemokininduzierte Migration von Leukozyten
Authors: Rink, Ina
Journal: (2016)

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