Figure 1 – Effect of Varying Primary Antibody Concentration:
Western blot optimization using R&D Systems rabbit anti-human/mouse/rat RSK1 affinity purified polyclonal antibody (Catalog # AF992). Human HeLa cells were solubilized in SDS gel buffer, and lysate from 1 x 105 cells per lane was resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.
A Dot Blot is a simple and quick assay that may be employed to determine if your antibodies and detection system are effective. Dot Blot may also be used to determine appropriate starting concentration of primary antibody for Western blot.
This protocol is intended to provide a set of initial conditions for analysis of cell lysates samples by Western blot. Further optimization may be required for individual samples or analytes. Follow manufacturer's protocols for specific reagents when applicable.
R&D Systems Quality Control laboratories use these Western blotting and immunostaining protocols to show that our polyclonal and monoclonal antibodies are specific for the proteins they were raised against and to determine the sensitivity of the antibody for its antigen.