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Fluorescein-5-maleimide *CAS 75350-46-8*
Maleimides are among the most frequently used reagents for thiol modification. In most proteins, the site of reaction is at cysteine residues that either are intrinsically present or resulted from reduction of cystines. Unlike iodoacetamides, maleimides do not react with histidine and methionine under physiological conditions. Fluorescein-5-maleimide is one of the most popular fluorescent dyes for thiol modifications of proteins along with 5-TMRIA.
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
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 | - | - |
Citations
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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)
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)
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Journal: ACS Biomaterials Science & Engineering (2016): 501--507
Authors: Wu, Xiaohui and Han, Zheng and Schur, Rebecca M and Lu, Zheng-Rong
Journal: ACS Biomaterials Science & Engineering (2016): 501--507
References
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