To maximize the signal to noise ratio, the correct dilutions of primary antibody and secondary antibody conjugate need to be empirically determined and depend on several factors including the membrane type, the substrate to be used, the blocking/diluent solution and the amount of target. WestVision™ Block and Diluent is intended to be used as supplied without further dilution.
Due to the high sensitivity provided by the WestVision™ Block and Diluent, lower concentration of primary antibody and secondary antibody conjugates may be required to achieve optimal results.
As a starting point for chemiluminescent detection, dilute the primary antibody to 0.1 to 1.0 ug/ml and dilute the secondary antibody conjugate to 0.02 – 0.2 ug/ml, in WestVision™ Block and Diluent.
As a starting point for chromogenic detection, dilute the primary antibody to 0.25 to 1.0 ug/ml and dilute the secondary antibody conjugate to 0.2 – 1.0 ug/ml in WestVision™ Block and Diluent.
IMPORTANT: Not all blocking solutions are appropriate for all Western blot assays. Blocking solutions should be tested for compatibility in a given assay.
Remove the membrane from the transfer device.
Rinse briefly with deionized (DI) H2O.
Add enough WestVision™ Block and Diluent to completely cover the membrane and incubate with agitation for 30 – 60 minutes.
Continue with your detection protocol using WestVision™ Block and Diluent to dilute primary antibody and detection reagents.