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By Pranveer Singh

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Pre-concentration The sole purpose of pre-concentration step is to concentrate the protein to very high levels (>100 mg/mL) within the dextran matrix, to drive the coupling reaction. Otherwise, higher concentrations of protein would need to be injected to get equivalent levels of coupling. An electrostatic interaction between the negatively charged carboxylated dextran matrix and positively charged protein drives the pre-concentration step. Protein is diluted into a buffer with a low ionic strength (to minimize charge screening) with a pH below its isoelectric point or pI (to give the protein net positive charge).

The bait protein and possibly the prey protein. 1. A TYPICAL EXPERIMENT A typical SPR experiment involves several discrete tasks. • • • • • • Prepare ligand and analyte. Select and insert a suitable sensor chip (CM 5 or others available). Immobilise the ligand and a control ligand to sensor surfaces. Inject analyte and a control analyte over sensor surfaces and record response. Regenerate surfaces if necessary. Analyse data. While the ligand and analyte could be almost any type of molecule, they are usually both proteins.

Mutants can be expressed as tagged proteins by transient transfection and then captured from crude tissue culture supernatant using an antibody to the tag, thus effectively purifying the mutant protein on the sensor surface. SPR helps to visualise the capture of proteins from crude mixtures onto the sensor surface. This elucidates the effect of the mutation on the binding properties (affinity, kinetics, and even thermodynamics) of the immobilised protein. This is the only practical way of quantifying the effect of Applications 33 mutations on the thermodynamics and kinetics of weak protein/ligand interactions [38].

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