iFluor® 625 succinimidyl ester
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Storage||Freeze (< -15 °C); Minimize light exposure|
|iFluor® 625 maleimide|
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
Mix 100 µL of a reaction buffer (e.g., 1 M sodium bicarbonate solution or 1 M phosphate buffer with pH ~8.5 to 9.0) 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 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. The final protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.
Add anhydrous DMSO into the vial of iFluor® 625 SE 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 the freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.
SAMPLE EXPERIMENTAL PROTOCOL
This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor® 625 SE. You might need further optimization for your particular proteins.
Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity, while the protein conjugates of low dye/protein ratio give reduced sensitivity.
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) 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 using a 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.
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 must be diluted with staining buffer, and aliquoted for multiple uses.
Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.
Common stock solution preparation
|0.1 mg||0.5 mg||1 mg||5 mg||10 mg|
|1 mM||87.566 µL||437.832 µL||875.664 µL||4.378 mL||8.757 mL|
|5 mM||17.513 µL||87.566 µL||175.133 µL||875.664 µL||1.751 mL|
|10 mM||8.757 µL||43.783 µL||87.566 µL||437.832 µL||875.664 µL|
|Mass (Calculate)||Molecular weight||Volume (Calculate)||Concentration (Calculate)||Moles|
|Name||Excitation (nm)||Emission (nm)||Extinction coefficient (cm -1 M -1)||Quantum yield||Correction Factor (260 nm)||Correction Factor (280 nm)|
|iFluor® 350 succinimidyl ester||345||450||200001||0.951||0.83||0.23|
|iFluor® 405 succinimidyl ester||403||427||370001||0.911||0.48||0.77|
|iFluor® 488 succinimidyl ester||491||516||750001||0.91||0.21||0.11|
|iFluor® 514 succinimidyl ester||511||527||750001||0.831||0.265||0.116|
|iFluor® 532 succinimidyl ester||537||560||900001||0.681||0.26||0.16|
|iFluor® 555 succinimidyl ester||557||570||1000001||0.641||0.23||0.14|
|iFluor® 594 succinimidyl ester||588||604||1800001||0.531||0.05||0.04|
|iFluor® 633 succinimidyl ester||640||654||2500001||0.291||0.062||0.044|
|iFluor® 647 succinimidyl ester||656||670||2500001||0.251||0.03||0.03|
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