ReadiUse™ Preactivated PE Maleimide [Activated R-Phycoerythrin]

ReadiUse™ Preactivated PE Maleimide is prepared by reacting highly purified R-Phycoerythrin (PE) with SMCC. The NHS group of SMCC reacts with the lysine groups of PE, leaving maleimide groups available to react with free sulfhydryl groups on proteins to be conjugated. ReadiUse™ Preactivated PE Maleimide is highly purified to completely remove the unreacted SMCC, and lyophilized to a powder form. It is ready to use and will conjugate without further preparation upon mixing with sulfhydryl-containing target molecules such as reduced IgGs. This activated phycobiliprotein can be easily conjugated to antibodies and other proteins without the use of added chemical crosslinking agents. The prepared conjugates maintain the spectral characteristics of PE.

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Reduction of IgG

Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody reduction can be carried out in different buffers, such as MES, phosphate, or TRIS (pH range 6 to 8). For best results, antibody solutions should be >2 mg/mL. If less than 2 mg/mL, the antibody should be concentrated.
Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution, and mix well. Let the antibody solution stand at room temperature for 30 minutes without additional mixing to minimize the reoxidation of cysteines.
Purify the reduced antibody using a desalting column (Cat No. 60500) pre-equilibrated with 50 mM MES Buffer (pH 6.0-6.5) and 2 mM EDTA.
Measure the antibody concentration using a Nanodrop. (Con. (mg/mL) = A280nm/1.4). Note: Reduced antibodies are typically unstable. It is best to run the conjugation reaction immediately following purification.

Conjugate with ReadiUse™ Preactivated PE Maleimide

Reconstitute ReadiUse™ Preactivated PE Maleimide in ddH2O to make a 10 mg/mL solution. For Cat No. 2565 (1 mg) reconstitute in 100 µL of ddH2O. If using Cat No. 2566 (5 mg), reconstitute in 500 µL of ddH2O. Reconstituted ReadiUse™ Preactivated PE Maleimide solutions are stable at 4 °C for up to one week, protected from light.
Add the reduced antibody directly to the solution of ReadiUse™ Preactivated PE at a ratio of 250-300 µg PE/100 µg of reduced antibody.
Rotate the mixture for 60 to 120 minutes at room temperature.
Block any free sulfhydryl groups on the antibody. Prepare a fresh solution of 10 mg/mL N-Ethylmaleimide (NEM) in DMSO and add 3.4 µL per mg of antibody. Rotate for 20 minutes at room temperature.

Conjugate Purification

Purify the antibody/PE conjugate solution using size exclusion chromatography. Note: Typically the antibody/PE conjugate can be stored at 2-8 °C, protected from light, for up to 6 months. The best storage conditions for any particular conjugate must be determined by experimentation and depends on the stability of the antibody.

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (280 nm)
ReadiUse™ Preactivated APC Maleimide [Activated Allophycocyanin]	651	660	730000	0.195
ReadiUse™ Preactivated PerCP Maleimide	477	678	406000	0.22

Citations

View all 1 citations: Citation Explorer

Targeting pro-inflammatory T cells as a novel therapeutic approach to potentially resolve atherosclerosis in humans
Authors: Fan, Lin and Liu, Junwei and Hu, Wei and Chen, Zexin and Lan, Jie and Zhang, Tongtong and Zhang, Yang and Wu, Xianpeng and Zhong, Zhiwei and Zhang, Danyang and others,
Journal: Cell Research (2024): 1--21

References

View all 24 references: Citation Explorer

Difference between mitogen-stimulated B and T cells in nonspecific binding of R-phycoerythrin-conjugated antibodies.
Authors: Bohacova, Pavla and Kossl, Jan and Hajkova, Michaela and Hermankova, Barbora and Holan, Vladimir and Javorkova, Eliska
Journal: Journal of immunological methods (2021): 113013

Relationship between circulating tumor-associated autoantibodies and clinical outcomes in advanced-stage NSCLC patients receiving PD-1/-L1 directed immune checkpoint inhibition.
Authors: Tarhoni, Imad and Wakefield, Connor J and Kollipara, Revathi and Fidler, Mary Jo and Batus, Marta and Bonomi, Philip and Borgia, Jeffrey A
Journal: Journal of immunological methods (2021): 112956

Quantification and Imaging of Antigens on Cell Surface with Lipid-Encapsulated Fluorescent Nanodiamonds.
Authors: Hsieh, Feng-Jen and Chen, Yen-Wei and Hui, Yuen Yung and Lin, Chun-Hung and Chang, Huan-Cheng
Journal: Micromachines (2019)

Development of bead based multiplexed immunoassay for evaluation of midkine, syndecan-1, and ANGPTL4 in patient serum.
Authors: Tarhoni, Imad and Fhied, Cristina L and Pool, Mark and Liptay, Michael J and Bonomi, Philip and Seder, Christopher W and Borgia, Jeffrey A
Journal: Journal of immunoassay & immunochemistry (2018): 84-98

R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.
Authors: Kim, Myun Soo and Kim, Tae Sung
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2013): 452-60

Page updated on November 20, 2024

Ordering information

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Unit size	1 mg 5 mg
Catalog Number	25652566
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Physical properties

Solvent

Water

Spectral properties

Correction Factor (280 nm)	0.175
Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	574
Quantum yield	0.82

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure; Desiccated
UNSPSC	12171501