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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 prefered.
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 reduce 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 1.5 mg of SMCC activated APC (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 APC–antibody conjugate, with small amounts of free APC and antibody. Unconjugated APC 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.
Antibody Conjugate.
2.4. For protein concentration calculation, please refer to Appedix II, Characterization of APC-Antibody Conjugate.
3. Storage of conjugates
3.1. Store the APC-labeled antibody at concentration >0.5 mg/mL or add 1-10 mg/mL of BSA as a stabilizer.
3.2. Add preservatives (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. Cohen-Bazire, G. et al. Arch. Microbiol. 111, 225 (1977).
2. Brejc, K. et al. J. Mol Biol. 249, 424 (1995).
3. Sidler, W. et al. Physiol. Chem. 362, 611 (1981).
4. Oi, VT. et al. J. Cell Biol. 93, 981 (1982).
5. Kronick, MN and PD. Grossman PD Clin. Chem. 29, 1582 (1983).
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Contents/Specifications |
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The amount of APC 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. Cross-linked and SMCC-activated APC are also available as separate products.
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Notes |
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The AnaTag™ APC Labeling Kit provides a convenient way to conjugate antibodies to allophycocyanin (APC). APC belongs to the phycobiliproteins family of highly soluble and fluorescent proteins derived from cyanobacteria and eukaryotic algae.1-3 APC, an ultra-sensitive fluorescent tracer with high emission quantum yields, emits in the near-infrared fluorescence range. It therefore avoids interference from autofluorescence of cellular components and other biological materials. APC is significantly more sensitive than conventional organic fluorophores and has been used in applications such as flow cytometry, homogeneous FRET assay and immunoassays. 4, 5 The AnaTagTM APC Labeling Kit is a SH-reactive labeling kit. SMC leimide 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 APC and sulfhydryl group on the antibody form a covalent bond during conjugation. The AnaTagTM APC Labeling Kit contains a chemically cross-linked APC (CL-APC) that is more stable than the native APC, yet still retaining the original spectroscopic properties. 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™ APC Labeling Kit is optimized for use in the conjugation of allophycocyanin (APC) to antibodies. APC is an ultra-sensitive, near-infrared fluorescent tracer with a high quantum yield (Ex/Em = 650 nm/660 nm). APC- labeled antibodies are used in applications such as flow cytometry and immunofluorescence. The AnaTagTM APC Labeling Kit contains SH-reactive APC. SMCC modified allophycocyanin reacts with the thiol groups of target antibody without the need for additional activation, thus simplifying the conjugation protocol. AnaTagTM APC Labeling Kit contains a chemically cross-linked APC (CL-APC) that is much more stable than the native APC, but still retains the original spectroscopic properties.
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