1. Dissolve the protein to be modified at a concentration of 1–10 mg/ml in 0.1 M sodium phosphate, pH 7.4. NaCl may be added to this buffer if desired. For the modifi cation of keyhole limpet hemocyanin (KLH; Thermo Fisher) as described by Staros et al., 1986, include 0.9 M NaCl to maintain the solubility of this high-molecular-weight protein.If lower or higher concentrations of the protein are used, adjust the amounts of the other reactants as necessary to maintain the correct molar ratios.
2. Dissolve the molecule to be coupled in the same buffer used in step 1. For small molecules, add them to the reaction in at least a 10-fold molar excess over the amount of protein present. If possible, the molecule may be added directly to the protein solution in the appropriate excess. Alternatively, dissolve the molecule in the buffer at a higher concentration, and then add an aliquot of this stock solution to the protein solution.
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控制器 3. Add the solution prepared in step 2 to the protein solution to obtain at least a 10-fold molar excess of small molecule to protein.
4. Add EDC (Thermo Fisher) to the above solution to obtain at least a 10-fold molar exces
s of EDC over the amount of protein present. Alternatively, a 0.05–0.1 M EDC concentrationin the reaction usually works well. Also, add sulfo-NHS (Thermo Fisher) to the reaction to bring its final concentration to 5 mM. To make it easier to add the correct quantity of EDC or sulfo-NHS, higher concentration stock solutions may be prepared if they are dissolved and used immediately. Mix to dissolve. If this ratio of EDC/sulfo-NHS to peptide or protein results in precipitation, scale back the amount of addition until a soluble conjugate is obtained.
5. React for 2 hours at room temperature.
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6. Purify the conjugate by gel filtration or dialysis using the buffer of choice (for many conjugates 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4 is appropriate). If some turbidity has formed during the conjugation procedure, it may be removed by centrifugation or filtration. 在线测厚>
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