RRC-PRL: Coupling of Synthetic Peptide to Carrier Protein Using MBS

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  1. Dissolve 5.0 mg of KLH (Pierce-177600) in 0.5 ml of 0.01 M phosphate buffer, pH 7.0 [Note 1]. Or dissolve 20 mg of KLH in 2 ml of PB.
  2. Dissolve 3.0 mg of MBS (M-2786-sigma) [Note 2] in 200 µL DMF. Or 4.2 mg per 280 µL of DMF.
  3. Add 70 µL of MBS solution to 0.5 ml of KLH solution. Or add 280 µL of MBS to 2.0 ml of KLH solution.
  4. After stirring or rotating for 30 min at room temp, the KLH/MBS solution is passed through PD-10 column using 0.05 M PB, pH 6.0 as eluent [Note 3]. Collect the 3.5 ml of purified KLH/MBS and add to it 0.5 ml of water to make it 4.0 ml.
  5. Dissolve 5.0 mg of peptide in either 1.0 ml of 6 M guanidine-HCl/0.01 M phosphate buffer, pH 7.0 [Note 4] or 100 µL of DMF. To which rapidly add 1.0 ml of purified KLH/MBS. Shake rapidly and immediately add 11.0 µL of 2N NaOH [Note 5].
  6. Check the pH with pH paper. It should be 7.0 or 7.2. Too high pH or too low pH will stop the reaction between KLH/MBS and peptide. If needed, add immediately appropriate amount of 0.5 N HCl to change the pH.
  7. Stir or rotate the solution 3 hrs or overnight at 4 degrees. Afterwards lyophilize it.

Notes:

  1. PB 7.0 is made by adding 2.68 g of dibasic sodium phosphate to 1000 ml of d-I water and another addition of 380 µL of concentrated HCl (12.1 N) - confirm pH.
  2. MBS to be used is M2786. A less pure grade from Sigma will produce a pink color.
  3. PB 6.0 is made by adding 13.4 g of dibasic sodium phosphate to 1000 ml of d-I water and addition of 3800 µL of concentrated HCl - check pH.
  4. 2.87 g guanidine-HCl in 5.0 ml 0.01 M phosphate buffer.
  5. If DMF exceeds 100 µL, the solution may turn cloudy. Some peptides, even if 100 µL of DMF is used, will give cloudy appearance. The reaction can be kept longer for such peptides. Some peptides in DMF will be gelly. Gellyness will go away upon mixing PB and base followed by sonication. It is best to add DMF/peptide to KLH solution slowly rather than KLH to DMF/peptide.
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