iFluor® 860 maleimide
In vivo fluorescence imaging uses a sensitive camera to detect the fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the infrared (IR) region are generally preferred. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments aim to achieve high-resolution, multimodality, and lifetime-based in vivo fluorescence imaging. Our iFluor® 860 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 860 have excitation and emission in the IR range. iFluor® 860 dye emission is well separated from commonly used far-red fluorophores such as Cy5, Cy7, or allophycocyanin (APC), facilitating multicolor analysis. This fluorophore is also useful for small animal in-vivo imaging applications or other imaging applications requiring IR detection. iFluor® 860 maleimide is thiol-reactive and can be readily used to conjugate thiol-containing biomolecules.
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.
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.
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.
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:
1. iFluor™ 860 maleimide stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 860 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.
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 iFluor™ 860 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.
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 iFluor® 860 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 60.692 µL | 303.461 µL | 606.921 µL | 3.035 mL | 6.069 mL |
5 mM | 12.138 µL | 60.692 µL | 121.384 µL | 606.921 µL | 1.214 mL |
10 mM | 6.069 µL | 30.346 µL | 60.692 µL | 303.461 µL | 606.921 µL |
Molarity calculator
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Spectrum
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Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
iFluor® 350 maleimide | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
iFluor® 488 maleimide | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
iFluor® 555 maleimide | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
iFluor® 647 maleimide | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
iFluor® 680 maleimide | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
iFluor® 700 maleimide | 690 | 713 | 2200001 | 0.231 | 0.09 | 0.04 |
iFluor® 750 maleimide | 757 | 779 | 2750001 | 0.121 | 0.044 | 0.039 |
iFluor® 790 maleimide | 787 | 812 | 2500001 | 0.131 | 0.1 | 0.09 |
iFluor® 800 maleimide | 801 | 820 | 2500001 | 0.111 | 0.03 | 0.08 |
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References
View all 18 references: Citation Explorer
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Journal: J Immunol Methods (2007): 65
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Authors: Hama Y, Urano Y, Koyama Y, Kamiya M, Bernardo M, Paik RS, Shin IS, Paik CH, Choyke PL, Kobayashi H.
Journal: Cancer Res (2007): 2791
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Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis
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