Time Resolved Fluorescent Chelates

Introduction

Fluorescent isothiocyanato-activated (ITC-activated) Eu- and Tb- chelates are optimized for labelling proteins and peptides containing at least one primary aliphatic amino group.

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Labelling of Proteins

Eu- and Tb-chelates have an aromatic isothiocyanato group as a reactive arm. Isothiocyanato group reacts with primary aliphatic amino groups at alkaline pH.

1. Parameters of labelling reaction. Parameters of labelling reaction include protein concentration, pH, temperature,reaction time and molar excess of chelate over protein

2. Labelling. The recommended reaction concentrations for labelling proteins are pH 9.8, +4ºC and overnight incubation. Under these conditions, the following calculations are valid for labelling of a protein with an isoelectric point (pl) between 4 and 7.

Table 1. The effect of protein concentration on the percentage of chelate reacting with the protein. For example, if a protein (pl around 6, molecular weight 160 000) is reacted at a concentration of 5 mg/mL under the conditions described above, a 15-fold molar excess of chelate over protein would give a labelling degree of about 6 chelates per protein. If the protein to be labelled is not stable in the labelling conditions (+4ºC, pH 9.8, overnight incubation), it is possible to run a 4-hour reaction (+4ºC, pH 9.8) by increasing the molar excess of chelate over protein.

A suitable amount of chelate is three times higher for the 4 hour reaction to that for the overnight reaction. For example, if a protein (5 mg/mL in the labelling reaction) requires 15-fold molar exces of chelate during overnight reaction for the introduction of 6 chelates per protein, 45-fold molar excess of chelate is needed to obtain the same label incorporation during a 4-hour reaction. Suitable number of chelates coupled to a protein depends on the molecular weight (MW). When the MW of a protein is higher than 100 000, 4-10 chelates per protein is a good labelling yield. For proteins with a MW in the range of 30 000 – 70 000, the preferred number of coupled chelates is 2-6. Proteins with a MW less than 30 000 should be labelled with 1-2 Chelates. The given value may be higher for basic proteins (pl between 8 and 10).