agitation to reconstitute a 7 point 2-fold serial dilution curve by mixing 250 µl of top standard with 250 µl PBS 1% BSA and repeating with each standard down the curve. Following completion of the blocking step the samples can be added to the plate. 100 µl sample or standard (diluted to the appropriate concentration for each assay) was added to the appropriate wells following a detailed plate map. Plate was then incubated at room temperature for 2 h on the bench. Following this the plate was washed as above. To detect the protein of interest a biotinylated detection antibody was used to detect the protein and HRP conjugated avidin was added to bind to the biotinylated antibody. Firstly, the lyophilized antibody was reconstituted in 1 ml of PBS and diluted to 200 ng/ml in PBS 1% BSA 2% heat inactivated normal goat serum. 100 µl per well was added to the plate and incubated at room temperature for 2 h. The plate was then washed as above. To detect the bound antibody 100 µl of 1:200 dilution of the HRP-streptavidin was added to each well and incubated at room temperature for 20 minutes in the dark followed by repeating the washing step. 100 µl substrate solution (DY999) was then added and incubate at room temperature for 20 mins in the dark. Wells should go blue if protein is detected.
agitation to reconstitute a 7 point 2-fold serial dilution curve by mixing 250 µl of top standard with 250 µl PBS 1% BSA and repeating with each standard down the curve. Following completion of the blocking step the samples can be added to the plate. 100 µl sample or standard (diluted to the appropriate concentration for each assay) was added to the appropriate wells following a detailed plate map. Plate was then incubated at room temperature for 2 h on the bench. Following this the plate was washed as above. To detect the protein of interest a biotinylated detection antibody was used to detect the protein and HRP conjugated avidin was added to bind to the biotinylated antibody. Firstly, the lyophilized antibody was reconstituted in 1 ml of PBS and diluted to 200 ng/ml in PBS 1% BSA 2% heat inactivated normal goat serum. 100 µl per well was added to the plate and incubated at room temperature for 2 h. The plate was then washed as above. To detect the bound antibody 100 µl of 1:200 dilution of the HRP-streptavidin was added to each well and incubated at room temperature for 20 minutes in the dark followed by repeating the washing step. 100 µl substrate solution (DY999) was then added and incubate at room temperature for 20 mins in the dark. Wells should go blue if protein is detected.