iFluor® 500 succinimidyl ester
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Molecular weight | 981.12 |
Solvent | DMSO |
Excitation (nm) | 501 |
Emission (nm) | 520 |
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 |
Overview | SDSProtocol |
Molecular weight 981.12 | Excitation (nm) 501 | Emission (nm) 520 |
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
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® 500 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® 500 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.
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 (e.g., DOS > 6) 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 6-8 moles of iFluor® 500 SE to one mole of antibody. The following steps are used to determine the DOS of iFluor® 500 SE labeled proteins.
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.
For most spectrophotometers, the sample (from the column fractions) needs to be diluted with de-ionized water so that the O.D. 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 520 nm is the maximum absorption of iFluor® 500 SE. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.
You can calculate DOS using our tool by following this link: https://www.aatbio.com/tools/degree-of-labeling-calculator
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 101.924 µL | 509.622 µL | 1.019 mL | 5.096 mL | 10.192 mL |
5 mM | 20.385 µL | 101.924 µL | 203.849 µL | 1.019 mL | 2.038 mL |
10 mM | 10.192 µL | 50.962 µL | 101.924 µL | 509.622 µL | 1.019 mL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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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 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 | 587 | 603 | 2000001 | 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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References
Authors: Shih, Cheng-Wei and Chang, Chun-Hui
Journal: Brain structure & function (2023)
Authors: Rastogi, Simran and Rani, Komal and Rai, Sanskriti and Singh, Rishabh and Bharti, Prahalad Singh and Sharma, Vaibhav and Sahu, Jyoti and Kapoor, Vrinda and Vishwakarma, Poorvi and Garg, Sumit and Gholap, Shivajirao Lahu and Inampudi, Krishna Kishore and Modi, Gyan Prakash and Rani, Neerja and Tripathi, Madhavi and Srivastava, Achal and Rajan, Roopa and Nikolajeff, Fredrik and Kumar, Saroj
Journal: BMC medicine (2023): 335
Authors: Yang, Ruochen and Zhang, Geoff G Z and Zemlyanov, Dmitry Y and Purohit, Hitesh S and Taylor, Lynne S
Journal: Journal of pharmaceutical sciences (2023): 304-317
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Journal: Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] Internatio (2023): e13245
Authors: ElMaraashly, Aya Hassan and Tario, Joseph and Hillengass, Jens and Qian, You-Wen
Journal: American journal of clinical pathology (2023)
Authors: Kudryavtsev, Igor and Zinchenko, Yulia and Starshinova, Anna and Serebriakova, Maria and Malkova, Anna and Akisheva, Tatiana and Kudlay, Dmitriy and Glushkova, Anzhela and Yablonskiy, Piotr and Shoenfeld, Yehuda
Journal: Diagnostics (Basel, Switzerland) (2023)
Authors: Fogarty, Matthew J and Dasgupta, Debanjali and Khurram, Obaid U and Sieck, Gary C
Journal: Molecular and cellular neurosciences (2023): 103847
Authors: Jung, Seung-Ryoung and Kim, James and Vojtech, Lucia and Vaughan, Joshua C and Chiu, Daniel T
Journal: The journal of physical chemistry. B (2023): 2701-2707
Authors: Suzuki, Hideyuki and Thongbhubate, Kullathida and Muraoka, Madoka and Sasabu, Asuka
Journal: Enzyme and microbial technology (2023): 110139
Authors: Oka, Yoshimi
Journal: ACS omega (2023): 1109-1113