2-Mercaptoethanol → m-Maleimidobenzoyl-N-hydroxysuccinimide

Participants

Reaction Steps

To 500 μl of H2O were added 5 mg of the peptide and the pH was adjusted to 8.5 with NaOH, to obtain a final concentration of 10 mg/ml. Citraconic anhydride was diluted in H2O to a concentration of 10 mg/ml. 500 μl of the anhydride solution were added to the peptide solution 100 μl at a time with adjustment of the pH to 8.5 between each addition. The solution was then stirred constantly at room temperature for 1 hour. This was followed by the addition of 100 μl of 1M sodium phosphate buffer (pH 7.2) and then 900 μl of 100 mM sodium phosphate buffer (pH 7.2). Sulfo-MBS was diluted in H2O to a concentration of 25 mg/ml and 400 μl of this solution were added to the peptide solution to obtain an MBS concentration of about 5 mg/ml. This solution was stirred constantly at room temperature for 30 minutes. 6 μl of β-mercaptoethanol were added for a final β-mercaptoethanol concentration of 35 mM. The solution was stirred constantly at room temperature for 1 hour. KLH was dissolved in PBS at 3 mg/ml and 2.5 ml were added to the peptide solution. The solution was stirred constantly at room temperature for 3 hours and then dialysed against 1 litre of PBS, with three changes of the PBS. The final peptide concentration was about 1 mg/ml and the final KLH concentration was about 1.5 mg/ml.

Provenance & Evidence