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ICG Maleimide
Indocyanine green (ICG) is a cyanine dye used in medical diagnostics. It is used for determining cardiac output, hepatic function, and liver blood flow, and for ophthalmic angiography. It has a peak spectral absorption close to 800 nm. These infrared frequencies penetrate retinal layers, allowing ICG angiography to image deeper patterns of circulation than fluorescein angiography. ICG binds tightly to plasma proteins and becomes confined to the vascular system. ICG has a half-life of 150 to 180 seconds and is removed from circulation exclusively by the liver to bile juice. A recent study indicated ICG targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. This thiol-reactive ICG derivative is used to make ICG bioconjugates with antibodies and other biological molecules.
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. ICG maleimide stock solution (Solution B)
Add anhydrous DMSO into the vial of ICG 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 ICG 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 ICG 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 | 117.221 µL | 586.105 µL | 1.172 mL | 5.861 mL | 11.722 mL |
| 5 mM | 23.444 µL | 117.221 µL | 234.442 µL | 1.172 mL | 2.344 mL |
| 10 mM | 11.722 µL | 58.61 µL | 117.221 µL | 586.105 µL | 1.172 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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Spectrum
Citations
View all 5 citations: Citation Explorer
Comparison of Near-Infrared Imaging Agents Targeting the PTPmu Tumor Biomarker
Authors: Johansen, Mette L and Vincent, Jason and Rose, Marissa and Sloan, Andrew E and Brady-Kalnay, Susann M
Journal: Molecular Imaging and Biology (2023): 1--14
Authors: Johansen, Mette L and Vincent, Jason and Rose, Marissa and Sloan, Andrew E and Brady-Kalnay, Susann M
Journal: Molecular Imaging and Biology (2023): 1--14
Assessment of Lexiscan for Blood Brain Barrier disruption to facilitate Fluorescence brain imaging
Authors: Pak, Rebecca W and Le, Hanh and Valentine, Heather and Thorek, Daniel and Rahmim, Arman and Wong, Dean and Kang, Jin U
Journal: (2017): ATu3B--2
Authors: Pak, Rebecca W and Le, Hanh and Valentine, Heather and Thorek, Daniel and Rahmim, Arman and Wong, Dean and Kang, Jin U
Journal: (2017): ATu3B--2
Bioengineering of injectable encapsulated aggregates of pluripotent stem cells for therapy of myocardial infarction
Authors: Zhao, Shuting and Xu, Zhaobin and Wang, Hai and Reese, Benjamin E and Gushchina, Liubov V and Jiang, Meng and Agarwal, Pranay and Xu, Jiangsheng and Zhang, Mingjun and Shen, Rulong and others, undefined
Journal: Nature Communications (2016): 13306
Authors: Zhao, Shuting and Xu, Zhaobin and Wang, Hai and Reese, Benjamin E and Gushchina, Liubov V and Jiang, Meng and Agarwal, Pranay and Xu, Jiangsheng and Zhang, Mingjun and Shen, Rulong and others, undefined
Journal: Nature Communications (2016): 13306
Single-Layer MoS2 Nanosheets with Amplified Photoacoustic Effect for Highly Sensitive Photoacoustic Imaging of Orthotopic Brain Tumors
Authors: Chen, Jingqin and Liu, Chengbo and Hu, Dehong and Wang, Feng and Wu, Haiwei and Gong, Xiaojing and Liu, Xin and Song, Liang and Sheng, Zonghai and Zheng, Hairong
Journal: Advanced Functional Materials (2016)
Authors: Chen, Jingqin and Liu, Chengbo and Hu, Dehong and Wang, Feng and Wu, Haiwei and Gong, Xiaojing and Liu, Xin and Song, Liang and Sheng, Zonghai and Zheng, Hairong
Journal: Advanced Functional Materials (2016)
Deep Photoacoustic/Luminescence/Magnetic Resonance Multimodal Imaging in Living Subjects Using High-Efficiency Upconversion Nanocomposites
Authors: Liu, Yu and Kang, Ning and Lv, Jing and Zhou, Zijian and Zhao, Qingliang and Ma, Lingceng and Chen, Zhong and Ren, Lei and Nie, Liming
Journal: Advanced Materials (2016)
Authors: Liu, Yu and Kang, Ning and Lv, Jing and Zhou, Zijian and Zhao, Qingliang and Ma, Lingceng and Chen, Zhong and Ren, Lei and Nie, Liming
Journal: Advanced Materials (2016)
References
View all 193 references: Citation Explorer
Sentinel lymph node biopsy using intraoperative indocyanine green fluorescence imaging navigated with preoperative CT lymphography for superficial esophageal cancer
Authors: Yuasa Y, Seike J, Yoshida T, Takechi H, Yamai H, Yamamoto Y, Furukita Y, Goto M, Minato T, Nishino T, Inoue S, Fujiwara S, Tangoku A.
Journal: Ann Surg Oncol (2012): 486
Authors: Yuasa Y, Seike J, Yoshida T, Takechi H, Yamai H, Yamamoto Y, Furukita Y, Goto M, Minato T, Nishino T, Inoue S, Fujiwara S, Tangoku A.
Journal: Ann Surg Oncol (2012): 486
Indocyanine green angiography-guided photodynamic therapy for treatment of chronic central serous chorioretinopathy: a pilot study
Authors: Yannuzzi LA, Slakter JS, Gross NE, Spaide RF, Costa DL, Huang SJ, Klancnik JM, Jr., Aizman A.
Journal: Retina (2012): 288
Authors: Yannuzzi LA, Slakter JS, Gross NE, Spaide RF, Costa DL, Huang SJ, Klancnik JM, Jr., Aizman A.
Journal: Retina (2012): 288
Using indocyanine green fluorescent lymphography and lymphatic-venous anastomosis for cancer-related lymphedema
Authors: Mihara M, Murai N, Hayashi Y, Hara H, Iida T, Narushima M, Todokoro T, Uchida G, Yamamoto T, Koshima I.
Journal: Ann Vasc Surg (2012): 278 e1
Authors: Mihara M, Murai N, Hayashi Y, Hara H, Iida T, Narushima M, Todokoro T, Uchida G, Yamamoto T, Koshima I.
Journal: Ann Vasc Surg (2012): 278 e1
Management of peripheral polypoidal choroidal vasculopathy with intravitreal bevacizumab and indocyanine green angiography-guided laser photocoagulation
Authors: Rishi P, Das A, Sarate P, Rishi E.
Journal: Indian J Ophthalmol (2012): 60
Authors: Rishi P, Das A, Sarate P, Rishi E.
Journal: Indian J Ophthalmol (2012): 60
Synthesis and characterization of bovine serum albumin-coated nanocapsules loaded with indocyanine green as potential multifunctional nanoconstructs
Authors: Jung B, Anvari B.
Journal: Biotechnol Prog (2012): 533
Authors: Jung B, Anvari B.
Journal: Biotechnol Prog (2012): 533
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