logo
AAT Bioquest

iFluor® 830 succinimidyl ester

In vivo fluorescence imaging uses a sensitive camera to detect the fluorescence emission from fluorophores in whole-body living small animals. Fluorophores with long emissions at the infrared (IR) region are generally preferred to overcome photon attenuation in living tissue. 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 developments aim to achieve high-resolution, multimodality, and lifetime-based in vivo fluorescence imaging. Our iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 dye emission is a unique color for spectrum flow cytometry as it 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® 830 succinimidyl ester is amine-reactive and can be readily used to conjugate amine-containing biomolecules, particularly antibodies.

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

Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~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. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

iFluor™ 830 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 830 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 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™ 830 SE. 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
  1. 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.

  2. 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.

  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to 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 resin surface.
  4. 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.

Characterize the Desired Dye-Protein Conjugate

The Degree of Substitution (DOS) is the most important factor for characterizing dye-labeled protein. Proteins of lower DOS usually have weaker fluorescence intensity, but proteins of higher DOS tend to have reduced fluorescence too. The optimal DOS for most antibodies is recommended between 2 and 10 depending on the properties of dye and protein. For effective labeling, the degree of substitution should be controlled to have 2-3 moles of iFluor™ 830 SE to one mole of antibody. The following steps are used to determine the DOS of iFluor™ 830 SE labeled proteins.

Measure absorption

To measure the absorption spectrum of a dye-protein conjugate, it is recommended to keep the sample concentration in the range of 1-10 µM depending on the extinction coefficient of the dye.

Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 830 nm for iFluor™ 830 dyes)

For most spectrophotometers, the sample (from the column fractions) need be diluted with de-ionized water so that the OD values are in the range of 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein while 830 nm is the maximum absorption of iFluor™ 830 SE. To obtain accurate DOS, make sure that the conjugate is free of the 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 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58760320000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71673915000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester82285025000010.110.16
iFluor® 860 succinimidyl ester85387825000010.10.14
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74076422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 570 succinimidyl ester55757012000010.581--
iFluor® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520800001-0.2060.088
Show More (36)

References

View all 15 references: Citation Explorer
Deep learning for in vivo near-infrared imaging.
Authors: Ma, Zhuoran and Wang, Feifei and Wang, Weizhi and Zhong, Yeteng and Dai, Hongjie
Journal: Proceedings of the National Academy of Sciences of the United States of America (2021)
Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.
Authors: Wang, Jun and Zhang, Shuyan and Xu, Xinhui and Xing, Yujun and Li, Zongru and Wang, Jinke
Journal: ACS omega (2020): 13829-13839
Measuring Kidney Perfusion, pH, and Renal Clearance Consecutively Using MRI and Multispectral Optoacoustic Tomography.
Authors: Minhas, Atul S and Sharkey, Jack and Randtke, Edward A and Murray, Patricia and Wilm, Bettina and Pagel, Mark D and Poptani, Harish
Journal: Molecular imaging and biology (2020): 494-503
Evaluation of Targeting Efficiency of Joints with Anticollagen II Antibodies.
Authors: Lofchy, Laren A and Vu, Vivian P and Banda, Nirmal K and Ramirez, Joseline Ramos and Smith, Weston J and Gifford, Geoffrey and Gaikwad, Hanmant and Scheinman, Robert I and Simberg, Dmitri
Journal: Molecular pharmaceutics (2019): 2445-2451
Pharmacokinetic and Biodistribution Studies of HPMA Copolymer Conjugates in an Aseptic Implant Loosening Mouse Model.
Authors: Wei, Xin and Li, Fei and Zhao, Gang and Chhonker, Yashpal Singh and Averill, Christine and Galdamez, Josselyn and Purdue, P Edward and Wang, Xiaoyan and Fehringer, Edward V and Garvin, Kevin L and Goldring, Steven R and Alnouti, Yazen and Wang, Dong
Journal: Molecular pharmaceutics (2017): 1418-1428
Page updated on December 11, 2024

Ordering information

Price
Unit size
Catalog Number1384
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

1527.88

Solvent

DMSO

Spectral properties

Absorbance (nm)

830

Excitation (nm)

830

Emission (nm)

867

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
 iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 succinimidyl ester is the most convenient reactive form for preparing the desired iFluor® 830 conjugates as it specifically and readily reacts with amino groups.
 iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 succinimidyl ester is the most convenient reactive form for preparing the desired iFluor® 830 conjugates as it specifically and readily reacts with amino groups.
 iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 succinimidyl ester is the most convenient reactive form for preparing the desired iFluor® 830 conjugates as it specifically and readily reacts with amino groups.