Information
Home Algae R-PE Labeling Kit is an SH-reactive labeling kit. Modified R-PE has maleimide groups that can conveniently conjugate with the SH groups of antibodies. The total conjugation time including incubation takes approximately 3 hours.
With a broad range of excitation wavelengths, R-PE can be easily excited by two laser sources (488 nm & 561 nm), applied to most fluorescence detection instruments i.e. Flow Cytometry, Immunofluorescence Microscopy (IF), Microarray Assay. Home Algae's R-PE absorbance maxima are at 496, 545, and 565 nm, its significant stokes shifts with a resolvable emission peak at 578 nm
Sample of Protocol
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Add Modifier Solution into antibody
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Incubate the reaction at room temperature for 30 min
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Add Pre-activated R-PE and incubate 1 hour at room temperature with agitation and protect from light
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Add Quencher into the conjugation mixture and mix completely
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Incubate at room temperature and keep away from light for 30 min, the conjugation is complete.
R-PE AntibodyLabeling Kit
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Are phycobiliproteins stored in PBS only and in PBS with ammonium sulfate different?PBPs in different buffer solutions both exhibit good long-term stability, the form with ammonium sulfate requires more purification processes, such as dialysis or sulfate precipitation, to fully remove the ammonium sulfate prior to making tandem dyes and antibody conjugations. Home Algae Biotech offers phycobiliproteins stored in PBS only and PBS with ammonium sulfate buffer solutions.
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Is the conjugation reaction linking the R-PE/APC to free sulfhydryl groups on the antibody?Home Algae's R-PE & APC Labeling Kit are SH-reactive labeling kits. Crosslinked R-PE and APC have maleimide (-NH2) groups that easily react with sulfhydryl (-SH) groups of target antibody without the need for additional activation. The maleinide group of R-PE/APC and sulfhydryl group on the antibody form a covalent bond during conjugation. Users can also use crosslinked R-PE & APC to label other molecules that have free sulfhydryl groups.
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Do I need to remove the free R-PE from the R-PE-antibody conjugate?After the conjugation procedures, reaction mixture should contain mostly the R-PE-antibody conjugate, with small amounts of unconjugated R-PE and antibody. Unconjugated R-PE should not interfere with the assay result. However, purification is suggested for labeling up to 1 mg of antibody. Please contact us for more technical advice.
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Can I purchase large pack of labeling kit and separate them into small vials on my own for several reactions?Yes, all pack sizes of our labeling kits are scalable and can be separated into small packs based on your experiment requirements.
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What are the molecular weight of purified R-PE and APC?The molecular weight of purified R-PE is 240 kDa, and for purified APC is 105 kDa. They are both perfect for detecting cell surface antigens to further conduct cell screening and cell imaging. With high molecular weight, PE and APC won't penetrate into the cell sample even if cell membrane ruptured, which greatly lower the risk of false-positive reaction. It should be noted that the relatively high molecular weight of R-PE may preclude its use in procedures requiring good penetration into cells and tissues. It is predominantly intended for surface labeling of cells for flow cytometry.
Benefits of R-PE
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Ideal for multi-color fluorescent staining
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Can be excited by two laser sources, experiments can be performed without equipment upgrades
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Significant stokes shifts with resolvable emission spectra
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Ensure result accuracy
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Bright Index: Level 5 with high quantum yield
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It should be noted that the relatively high molecular weight of R-PE may preclude its use in procedures requiring good penetration into cells and tissues. It is predominantly intended for surface labeling of cells for flow cytometry.
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Stability
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Minimal fluorescence quenching
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