Custom Radiolabeling

Custom Radioiodination and Tritium Labeling

We radiolabel peptides, protein and small molecules for use in our radioligand binding, receptor occupancy and autoradiography assays.

For peptides and proteins, Iodine-125 [¹²⁵I] is introduced onto exposed tyrosine residues to give a high specific activity tracer, well suited to sensitive in vitro and in vivo work. Tritium is used for small molecules, where the labeled compound is chemically near-identical to the parent and ideal for receptor binding studies.

Example HPLC Chronogram

Preparative reverse-phase HPLC of [¹²⁵I]angiotensin II on a C18 column (35% to 80% methanol, 0.1% TFA, 0.4 mL/min). The unlabeled peptide, seen by UV at 214 nm, elutes at about 15.5 minutes. We provide the radiochromatogram for every preparation..

Labeling Strategies

Iodogen® Radioiodination Reagent 

Iodogen is the method we use most. The oxidant is coated on the reaction vessel rather than dissolved with your protein, so the molecule is never exposed to oxidant in solution, and the reaction is stopped simply by removing it from the tube. It is fast, gentle, and tolerant of amine and azide-containing buffers. It labels tyrosine, and to a lesser extent histidine, residues across a wide range of proteins, antibodies and peptides, which makes it our preferred choice for most targets.

Lactoperoxidase Radioiodination 

Lactoperoxidase oxidises iodide-[125I] to its reactive form using hydrogen peroxide. It is the mildest of the direct methods, which makes it our preferred route for flexible or methionine-containing peptides, where stronger oxidants would risk oxidising sensitive residues.

Reaction with [¹²⁵I]Bolton-Hunter Reagent  

When a protein has no accessible tyrosine, or when iodinating a tyrosine would disturb the binding interface, we conjugate the [¹²⁵I]Bolton-Hunter reagent to a primary amine instead. The radioiodinated phenolic group attaches through a lysine side chain or the N-terminus. The trade-off is that a lysine is modified, so we check first that the modified residue is not itself required for binding.

Tritium Labeling   

We run tritium custom synthesis on small molicules through our specialist partner, Novandi Chemistry AB. Small molecules are labelled with tritium at specific activities sufficient for most receptor binding assays. A suitable precursor may need to be provided.

Choosing the Right Route

Purification

Labeled material is purified by reverse-phase HPLC for peptides and small molecules, or by size-exclusion chromatography for antibodies and proteins. For iodinated peptides, the radioactive product resolves from unlabeled starting material as a single peak, with identity supported by the expected retention shift and, where needed, co-injection of standards. You receive the radiochromatogram for your preparation.

We Confirm Target Binding is Retained

A label is only useful if the tracer behaves like the parent, so we validate this in house. Saturation binding using the new tracer determines its K_d and B_max, so its affinity and receptor density are known before any study begins. The validated tracer then goes straight into assays performed on-site, such as radioligand binding, and autoradiography. You will receive the binding data and the study results.

What We Need from You

• For peptides and proteins, 0.2 to 0.5 mg of the unlabeled peptide.
• For small molicules (tritium labeling), please inquire.

A short conversation is usually enough to recommend the best method for your desired tracer. Get in touch today to ask one of our scientists directly.

Gifford Bioscience is a full-service receptor pharmacology CRO. We design assays collaboratively, troubleshoot difficult targets, and label, validate and deploy tracers in-house. Tell us what you want to label and the assay you have in mind, and we will recommend and walk you through the best approach for your tracer.