Protein Protocols Protein Protocols

The Bolton and Hunter Method 969
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The Bolton and Hunter Method for Radiolabeling Protein
Graham S. Bailey
1. Introduction
This is an indirect method in which an acylating reagent (N-succinimidyl-3[4-hydroxyphenyl]propionate,
the Bolton and Hunter reagent), commercially available in a
radioiodinated form, is covalently coupled to the protein to be labeled (1). The [ 125 I]
Bolton and Hunter reagent reacts mostly with the side-chain amino groups of lysine
residues to produce the labeled protein. It is the method of choice for radiolabeling
proteins that lack tyrosine and histidine residues, or where reaction at those residues
affects biological activity. It is particularly suitable for proteins that are sensitive to the
oxidative procedures employed in other methods (see Chapters 132, 133, and 135).
2. Materials
1. [ 125 I] Bolton and Hunter reagent: 37 MBq (1 mCi), concentration 185 MBq/mL (5 mCi/mL)
(see Note 1).
2. Buffer A: 0.1 M sodium borate buffer, pH 8.5.
3. Buffer B: 0.2 M glycine in 0.1 M sodium borate buffer, pH 8.5.
4. Buffer C: 0.05 M sodium phosphate buffer containing 0.25% gelatin.
5. Protein to be iodinated: A 0.5–2.5 mg/mL solution is made in buffer A.
It is essential that none of the solutions contain sodium azide or substances with free
thiol or amino groups (apart from the protein to be labeled), since the Bolton and Hunter
reagent will react with those compounds.
3. Method
1. The [ 125 I] Bolton and Hunter reagent (0.2 mL) is added to a small glass test tube (1 × 5.5 cm)
and is evaporated to dryness under a stream of dry nitrogen.
2. All reactants are cooled in iced water (see Note 2).
3. The protein to be iodinated (10 µL) is added, and the tube is gently shaken for 15 min
(see Note 2).
4. Buffer B (0.5 mL) is added (see Note 3). The solution is mixed and allowed to stand for 5 min.
5. Buffer C (0.5 mL) is added with mixing (see Note 4). The resultant solution is then ready
for purification.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
969