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ReadiUse™ Preactivated APC-AF700 Maleimide

Ordering information
Price ()
Catalog Number2720
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weightN/A
SolventWater
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageRefrigerated (2-8 °C); Minimize light exposure
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OverviewpdfSDSpdfProtocol


Molecular weight
N/A
APC-AF700 is a popular tandem color used in flow cytometry. Its primary absorption peak is at 651 nm with emission peak at ~700 nm. AAT Bioquest offers this preactivated APC-AF700 to facilitate the APC-AF700 tandem conjugations to reduced antibodies and other biomolecules that contain a thiol group. Our preactivated APC-AF700 maleimide is prepared from the commonly used crosslinker SMCC, and ready to conjugate. Allophycocyanin (APC) is a phycobiliprotein isolated from Spirulina sp., a blue-green alga. Like other phycobiliproteins, APC is fluorescent, with an extremely high absorptivity and a high quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without altering its spectral characteristics.

Example protocol


SAMPLE EXPERIMENTAL PROTOCOL

Reduction of Antibody
  1. Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water.  Antibody solutions should be at 2 mg/mL or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 2 mg/mL.
  2. 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 temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
  3. Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA. (Desalting column:  https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500)
  4. Measure the Antibody concentration with Nanodrop. (Con. (mg/ml)= A280nm/1.4) Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification. 

Conjugate with ReadiUse™ Preactivated APC-AF700 Maleimide
  1. Reconstitute ReadiUse™ Preactivated APC-AF700 Maleimide in 100 µL ddH2O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated APC-AF700 Maleimide are stable at 4 °C for one week, please kept it from light.
  2. Add reduced antibody to pre-activated APC-AF700 directly to at the ratio of 130 µg APC-AF700 /100 µg reduced antibody.
  3. Rotate the mixture for 60-120 mins at room temperature.
  4. After 60 minutes, block the free sulfhydryls on the antibody.
  5. Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature. 

Purification
  1. Antibody/APC-AF700 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/APC-AF700 conjugate solution is recommended to be stored at 2~8 °C and kept from light. 

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated APC-Cy7 Maleimide754779700000

References


View all 8 references: Citation Explorer
Bivalent EGFR-Targeting DARPin-MMAE Conjugates.
Authors: Karsten, Lennard and Janson, Nils and Le Joncour, Vadim and Alam, Sarfaraz and Müller, Benjamin and Tanjore Ramanathan, Jayendrakishore and Laakkonen, Pirjo and Sewald, Norbert and Müller, Kristian M
Journal: International journal of molecular sciences (2022)
Detecting and predicting neutralization of alemtuzumab responses in MS.
Authors: Saxena, Gauri and Moore, James M and Jones, Meleri and Pryce, Gareth and Ali, Liaqat and Leisegang, Georgia R and Vijay, Vivek and Loveless, Samantha and Robertson, Neil P and Schmierer, Klaus and Giovannoni, Gavin and Gnananpavan, Sharmilee and Baker, David and Tallantyre, Emma C and Kang, Angray S
Journal: Neurology(R) neuroimmunology & neuroinflammation (2020)
Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.
Authors: Bennett, Kristen and Sadler, Natalie C and Wright, Aaron T and Yeager, Chris and Hyman, Michael R
Journal: Applied and environmental microbiology (2016): 2270-2279
Pharmacokinetic Profile and Acute Toxicological Properties of a Novel Radiosensitizer Cytosine-Phosphate-Guanosine Oligodeoxynucleotide 107 in Mice Following Intravenous and Orthotopic Administration.
Authors: Cen, Yanyan and Li, Xiaoli and Yin, Zhiwei and Yan, Zifei and Liu, Dan and Peng, Wei and Pan, Feng and Zhou, Hong
Journal: Nucleic acid therapeutics (2015): 254-65
Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.
Authors: Li, Hsing-Hui and Huang, Zi-Yu and Ye, Shih-Png and Lu, Chi-Yu and Cheng, Pai-Chiao and Chen, Shu-Hwa and Chen, Chii-Shiarng
Journal: PloS one (2014): e85119
Selective 351 nm photodissociation of cysteine-containing peptides for discrimination of antigen-binding regions of IgG fragments in bottom-up liquid chromatography-tandem mass spectrometry workflows.
Authors: Cotham, Victoria C and Wine, Yariv and Brodbelt, Jennifer S
Journal: Analytical chemistry (2013): 5577-85
Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone.
Authors: Hirota, Morihiko and Motoyama, Akira and Suzuki, Mie and Yanagi, Masashi and Kitagaki, Masato and Kouzuki, Hirokazu and Hagino, Shigenobu and Itagaki, Hiroshi and Sasa, Hitoshi and Kagatani, Saori and Aiba, Setsuya
Journal: The Journal of toxicological sciences (2010): 871-9
Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays.
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Journal: Analytical chemistry (2007): 6312-8