Serum resistant bis-sulfhydryl specific biotinylation reagent

A method for use of a biotinylation agent for biotinylation of a protein or peptide containing at least one disulfide bond comprising:

• reacting the biotinylation agent according to formula (I)
• With
• R1=alkyl, hydroxyalkyl or carboxyalkyl residue comprising 1 to 50 carbon atoms
• R2=alkyl, glycol, amine or peptide reside with 1 to 50 carbon atoms
• R3=Aryl, heteroaryl, with 3 to 50 carbon atoms
• R4, R5=Aryl, hetero-aryl, Alkyl with 3 to 50 carbon atoms
• reducing at least one disulfide bond of the protein or peptide;
• reacting the resulting free thiols with a biotinylation reagent according to formula (I) in a suitable buffer (e.g., PBS, pH 6-8) at a temperature between 4° C. and 37° C. for 0.5-18 hours, thereby re-bridging the original disulfide via a three-carbon linker and attaching one biotin moiety per modified disulfide;
• and
• optionally removing excess reagent and/or purifying the biotinylated conjugate.