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Protocol |
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1. Preparing the antibody for conjugation
1.1. Adjust your antibody concentration to 2-10 mg/mL. A higher protein concentration is preferred.
Note 1: The protein can be dissolved in phosphate, carbonate, borate, Tris or MOPS buffer, pH 6.0-8.0, without oxidizing reagents, protein stabilizers (e.g. BSA) or other proteins.
Note 2: The conjugation efficiency is poor when the concentration of protein is less than 2 mg/mL. You may concentrate the protein solution using a speedvac or a centrifugal filter (Millipore, Cat# 42407).
1.2. Add 20 μl of DTT solution (Component B) per mL of IgG solution. Mix the reagents gently.
1.3. Incubate the reaction in a tightly capped tube at room temperature for 30 min without agitation.
1.4. If the reaction mixture is less than 120 μL, use a spin column (Component C) to desalt the reduced antibody (refer to Appendix I. Spin Column Procedures). If the reaction mixture is more than 120 μL, use a gravity column (Component F) to desalt the reduced antibody (refer to Appendix I. Gravity Column Procedures).
1.5. Calculate the concentration of the eluted protein based on absorption at 280 nm. For antibody solution with a small volume, you may estimate the protein concentration by the amount used at the beginning of the reaction and the volume of the eluent. If the concentration is less than 2 mg/mL, it is necessary to concentrate the antibody solution to >2 mg/mL for better yield in the conjugation reaction.
Note 1: To confirm the fractions containing the antibody, use Dye Reagent Concentrate (Bio-Rad Protein Assay, Cat#500-0006)
Note 2: Antibody solution can be concentrated by using a speedvac or a centrifugal filter (Millipore, Cat# 42407).
2. Performing the conjugation reaction
2.1. Add 3 mg of SMCC activated B-PE (Component A) per mg of reduced IgG. Incubate 1 hour at room temperature with agitation and protect from light.
2.2. Block excess free thiols
- Add 10 μL of DMSO (Component G) into one vial of NEM (Component H).
- Add 1 μL of NEM solution per 100 μL of conjugation mixture from Step 2.1 and mix completely. Incubate at room temperature for 30 min and keep away from light.
2.3. Conjugation is complete. The reaction mixture should contain mostly the B-PE–antibody conjugate, with small amounts of free B-PE and antibody. Unconjugated B-PE should not interfere with the assay. If purification is necessary, use a Bio-gel A 1.5 m (Bio-rad Cat# 151-0450) or an affinity column for IgG.
2.4. For protein concentration calculation, please refer to Appendix II. Characterization of B-PE-Antibody Conjugate.
3. Storage of conjugates
3.1. Store the B-PE-labeled antibody at concentration > 0.5 mg/mL or add 1-10 mg/mL of BSA as a stabilizer.
3.2. Add preservative (e.g. 0.01% sodium azide).
3.3. Store the conjugate solution at 4°C in the dark for two months. DO NOT FREEZE.
3.4. If there is aggregation in the conjugate solution, centrifuge briefly for 30 sec and use the supernatant only.
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References |
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1. Glazer, AN et al. J.Biol.Chem. 252, 32-42 (1977).
2. Lundell, DJ et al. J.Biol.Chem. 259, 5472-5480 (1984).
3. Oi, VT. et al. J. Cell Biol. 93, 981 (1982).
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Contents/Specifications |
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The amount of B-PE supplied in this kit is sufficient for labeling up to 1 mg of antibody. The kit provides all reagents, purification columns and a detailed step-by-step protocol. Native and SMCC-activated B-PE are also available as separate products
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Notes |
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The AnaTag™ B-PE Labeling Kit provides a convenient way to conjugate antibodies to B-phycoerythrin (B-PE). B-PE belongs to the phycobiliproteins family of highly soluble and fluorescent proteins derived from cyanobacteria and eukaryotic algae.1 B-PE is made of α, β and γ subunits and is present as (αβ)6γ. The absorption bands peak at 545 nm (εM =2.41 X 106 M-1cm-1) and 563 (εM =2.33 X 106 M-1cm-1).1,2 B-PE and the closely related R-PE are significantly more sensitive and photostable than conventional organic fluorophores.3 B-PE labeled primary and secondary antibody have been widely used in applications such as flow cytometry, live cell staining, and multi-color immunofluorescent staining. The AnaTagTM B-PE Labeling Kit is a SH-reactive labeling kit. SMCC modified B-phycoerythrin has maleimide groups that easily react with SH groups of a target antibody without the need for an additional activation, thus simplifying the conjugation protocol. The maleimide group of B-PE and sulfhydryl group on the antibody form a covalent bond during conjugation. The kit provides all the reagents, purification columns needed to label up to 1 mg of antibody, as well as a detailed step-by-step protocol
The AnaTag™ B-PE Labeling Kit is optimized for use in the conjugation of B-Phycoerythrin (B-PE) to antibodies. B-PE, a fluorescent protein from phycobiliprotein family, has a primary absorption peak at 545 nm with a secondary peak at 563 nm and maximum emission at 578 nm. B-PE and the closely related R-PE are the most intensely fluorescent phycobiliproteins having orange fluorescence. They are significantly brighter and more photostable than conventional organic fluorophores. B-PE labeled primary and secondary antibodies are used in applications such as flow cytometry and multi-color immunofluorescent staining. The AnaTagTM B-PE Labeling Kit contains SH-reactive B-PE. SMCC modified B-PE reacts with the thiol groups of target antibody without the need for additional activation, thus simplifying the conjugation protocol.
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