ReadiUse™ Preactivated PE Maleimide [Activated R-Phycoerythrin]

Flow cytometry analysis of whole blood cells stained with CD8 (SK1)-PE conjugate prepared with ReadiUse™ Preactivated PE Maleimide (Cat No. 2565). The fluorescence signal was monitored using an Aurora flow cytometer in the B4-A channel.

Flow cytometry analysis of whole blood stained with PE anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE specific B4-A channel.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Solvent

Water

Spectral properties

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

Overview SDS Protocol

Extinction coefficient (cm ^-1 M ^-1)

1960000

Excitation (nm)

565

Emission (nm)

574

Quantum yield

0.82

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

Open in Advanced Spectrum Viewer

Spectral properties

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

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated APC Maleimide [Activated Allophycocyanin]	651	660	700000
ReadiUse™ Preactivated PerCP Maleimide	482	678	350000

Images

Figure 1. Flow cytometry analysis of whole blood cells stained with CD8 (SK1)-PE conjugate prepared with ReadiUse™ Preactivated PE Maleimide (Cat No. 2565). The fluorescence signal was monitored using an Aurora flow cytometer in the B4-A channel.

Figure 2. Flow cytometry analysis of whole blood stained with PE anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE specific B4-A channel.

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

HLA typing using bead-based methods.
Authors: Trajanoski, Daniel and Fidler, Samantha J
Journal: Methods in molecular biology (Clifton, N.J.) (2012): 47-65

Microarray Beads for Identifying Blood Group Single Nucleotide Polymorphisms.
Authors: Drago, Francesca and Karpasitou, Katerina and Poli, Francesca
Journal: Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie (2009): 157-160

The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2.
Authors: Kawasaki, Norihito and Ichikawa, Yoko and Matsuo, Ichiro and Totani, Kiichiro and Matsumoto, Naoki and Ito, Yukishige and Yamamoto, Kazuo
Journal: Blood (2008): 1972-9

[Determination of platelet-associate autoantibodies against platelet-specific receptors by cytometric bead array and its clinical application in idiopathic thrombocytopenic purpura].
Authors: Liu, Xin-Guang and Wang, Yan-Ming and Hou, Ming and Zhang, Ti and Zhu, Yuan-Yuan and Peng, Jun
Journal: Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi (2008): 175-8

In vitro evaluation of adenosine 5'-monophosphate as an imaging agent of tumor metabolism.
Authors: Cho, Steve Y and Polster, Josh and Engles, James M and Hilton, John and Abraham, Edward H and Wahl, Richard L
Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (2006): 837-45