ReadiLink™ BSA Conjugation Kit
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The following protocol is a general protocol for a wide variety of haptens. Optimize the protocol accordingly for the conjugation efficiencies upon the size and structure of your hapten. Using a molar excess of hapten over carrier protein ensures efficient conjugation. In general, a reaction with equal mass amounts of hapten and carrier protein will achieve sufficient molar excess.
1. Prepare BSA-Hepten Conjugation:
1.1 Add 200 mL of ddH2O into the vial of BSA (Component A) to make a 10 mg/mL solution.
1.2 Dissolve up to 2 mg hapten in 450 mL Conjugation Buffer (Component B).
Note: Some haptens might have limited solubility, use DMSO (< 30% in the final conjugation solution) to dissolve it first. Higher concentration of DMSO might irreversibly denature the carrier protein.
1.4 Add the Hapten-BSA solution (from Step 1.3) into one vial of EDC (10mg), dissolve it by gentle mixing. Incubate at room temperature for 2 hours.
1.5 Purify the conjugate by desalting to remove non-reacted crosslinker and protein preservative (e.g., sodium azide).
2. Purify BSA-Hepten conjugate:
2.1 Thaw 1 bottle of Purification Buffer (Component D) to room temperature before use. Unused buffer can be stored at 4 °C for 1 week.
2.2 Twist off the bottom closure of the desalting column (Component E), and loosen the cap. Place the column in a collection tube.
2.3 Centrifuge the column at 1,000g for 2 minutes to remove the storage solution.
2.4 Remove the cap and slowly add 1 mL of purification buffer to the column. Centrifuge at 1,000g for 2 minutes, remove the buffer. Repeat this step for 3 additional times, discarding the buffer from the collection tube.
2.5 Place the column to a new collection tube, and gently apply the sample into the center of the compact resin bed.
2.6 Centrifuge the column at 1,000g for 2 minutes to collect the sample.
2.7 The Hapten-BSA conjugate can now be used for immunization. If the Hapten-BSA conjugate is to be stored for more than a few days, sterile filter the conjugate, and store at 4 °C or - 20 °C.
Note 1: If the conjugate is to be used within one week, PBS may be used for purification. If the conjugate will be frozen, use the purification buffer salts (Component D) for purification.
Note 2: If DMSO is used in the conjugation, prepare the purification buffer salts with the same percentage of DMSO used for conjugation. This will minimize the precipitation in the column during desalting.
Note 3: If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify the supernatant. Combine the precipitate and the purified conjugate.
|References & Citations||
1. Zu Y, Zhang Y, Zhao X, Zhang Q, Liu Y, Jiang R. (2009) Optimization of the preparation process of vinblastine sulfate (VBLS)-loaded folate-conjugated bovine serum albumin (BSA) nanoparticles for tumor-targeted drug delivery using response surface methodology (RSM). Int J Nanomedicine, 4, 321.
2. Enomoto H, Li CP, Morizane K, Ibrahim HR, Sugimoto Y, Ohki S, Ohtomo H, Aoki T. (2008) Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate. J Food Sci, 73, C84.
3. Ledesma-Osuna AI, Ramos-Clamont G, Vazquez-Moreno L. (2008) Characterization of bovine serum albumin glycated with glucose, galactose and lactose. Acta Biochim Pol, 55, 491.
4. Wang JH, Wang HQ, Zhang HL, Li XQ, Hua XF, Cao YC, Huang ZL, Zhao YD. (2007) Purification of denatured bovine serum albumin coated CdTe quantum dots for sensitive detection of silver(I) ions. Anal Bioanal Chem, 388, 969.
5. Zheng C, Bi J, Ma G, Su Z. (2007) Polyethylene glycol improves conjugation of bovine hemoglobin and human serum albumin in a controlled ratio. Artif Cells Blood Substit Immobil Biotechnol, 35, 568.
6. Tattini V, Jr., Parra DF, Polakiewicz B, Pitombo RN. (2005) Effect of lyophilization on the structure and phase changes of PEGylated-bovine serum albumin. Int J Pharm, 304, 124.
7. Burkin AA, Kononenko GP, Soboleva NA. (2003) [Products of spontaneous conjugation of aflatoxins with bovine serum albumin: immunochemical properties]. Prikl Biokhim Mikrobiol, 39, 228.
8. Mao SJ, Hou SX, Zhang LK, Jin H, Bi YQ, Jiang B. (2003) [Preparation of bovine serum albumin nanoparticles surface-modified with glycyrrhizin]. Yao Xue Xue Bao, 38, 787.
9. Conlan JW, Shen H, Webb A, Perry MB. (2002) Mice vaccinated with the O-antigen of Francisella tularensis LVS lipopolysaccharide conjugated to bovine serum albumin develop varying degrees of protective immunity against systemic or aerosol challenge with virulent type A and type B strains of the pathogen. Vaccine, 20, 3465.
10. Nakamura M, Oba Y, Mori T, Sato K, Ishida Y, Matsuda T, Nakamura H. (2002) Generation of polyclonal antibody against mu-conotoxin GIIIA using an immunogen of [Cys(5)]mu-conotoxin GIIIA site-specifically conjugated with bovine serum albumin. Biochem Biophys Res Commun, 290, 1037.