Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)

Coat the corresponding wells of a 96-well plate (Nunc-Immuno MicroWell 96 well solid plate, MERCK) with 100µL of TBS-C containing 10 VLDLR ectodomain at 4°C.

Wash the plate once with TBS-C.

Add Blocking solution and incubate the plate for 2h 0m 0s at Room temperature (25 ºC). The wells without receptor are now coated with BSA.

Remove Blocking solution and apply a series of increasing concentrations of ligand diluted in Blocking solution, each in a final volume of 100µL. Each ligand concentration should be added to one well with immobilized VLDLR and one well coated with BSA (Blocking solution) for ligand background binding. One well with VLDLR and one well coated with BSA should be incubated without ligand to determine the general plate background signal.

Incubate 1h 0m 0s at Room temperature (25°C).

Wash the plate three times with Blocking solution.

Add the corresponding primary antibodies diluted 1/100 in Blocking solution and incubate 1h 0m 0s at Room temperature (25°C).

Wash the plate three times with TBS-C Blocking solution.

Add the corresponding secondary antibody (horseradish peroxidase (HRP) conjugated) diluted 1/10,000 in Blocking solution and incubate 1h 0m 0s at Room temperature (25°C).

Wash the plate three times with TBS-C Blocking solution.

Add 100µL per well of the HRP substrate 1-Step Ultra TMB ELISA Substrate Solutions (Thermo Fisher Scientific, 34028) to develop the plate and incubate until the desired color develops.

Add 100µL per well of quenching solution to stop the reaction.

Measure absorbance at 450 nm.