General ELISA Procedures

A detailed protocol is shipped with every Novex® ELISA kit. The general ELISA protocol, however, is quite simple and is shown in Figure 10 in schematic form. The procedure for Chemi ELISA kits is similar, except the readout is chemiluminescent instead of colorimetric.

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The assay procedure for phosphoELISA™ kits is similar to our general ELISA procedure with only a few minor differences (Figure 1). The detection antibody for ELISA kits is biotin-labeled and is followed by incubation with streptavidin-HRP, whereas the detection antibody for phosphoELISA™ kits is a rabbit polyclonal antibody and is followed by incubation with HRP-labeled anti–rabbit IgG.
 

 

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  Figure 1   Figure 2
elisa procedure
 
Figure 1. Fast and easy, 4-hour Novex® ELISA kit protocol. Capture antibodies are precoated on the bottom of a 96-well plate. The sample or standard is added to the wells and incubated to allow target proteins to bind. The wells are then washed to remove unbound material, and the detection antibody is added and incubated to form a sandwich around the protein of interest. HRP conjugate is then added and incubated to allow binding via a biotin–streptavidin interaction. Next, the chromogen substrate for HRP is added, and the subsequent enzymatic reaction turns the solution blue. Finally, the reaction is stopped, turning the solution yellow in proportion to the amount of target protein in the sample. Absorbance is read in a microplate reader at 450 nm.
 
Figure 2. General schematic of phosphoELISA™ protocol. Capture antibodies are precoated on the bottom of a 96-well plate. The cell extract or standard is added to the wells and incubated to allow target proteins to bind. The wells are then washed to remove unbound material, and the detection antibody is added and incubated to form a sandwich around the protein of interest. HRP anti-rabbit antibody is then added and incubated to allow binding to the rabbit-derived detection antibody. Next, the chromogen substrate for HRP is added, and the subsequent enzymatic reaction turns the solution blue. Finally, the reaction is stopped, turning the solution yellow in proportion to the amount of target protein in the sample. Absorbance is read in a microplate reader at 450 nm.