Additional Information about Protein Detection Methods
Our Western Blot Service is a highly sensitive low throughput method for protein identification via antigen-specific antibodies and molecular weight.
In the first step, the proteins are solubilized via a detergent from tissue or cells. Next, the proteins are separated by their molecular size via SDS-PAGE before being transferred onto a membrane. The proteins are detected by specific antibodies labeled with fluorochromes quantified with a near-infrared imager.
The more sensitive enhanced chemiluminescence (ECL)-based imaging can be used as an alternative detection method.
ELISA is well established at Reaction Biology for quantification of proteins in a high-throughput format.
The target protein can be quantified from liquid samples such as blood plasma or cell culture medium or solid samples including tissues or cultured cells. After lysis, the lysate is applied to multi-well plates prepared with target-specific capture antibodies immobilized on the plastic surface. The antibody captures the target proteins which in turn are detected by target-specific detection antibodies that are labeled with a fluorochrome for quantification.
AlphaLISA and AlphaScreen are homogenous assay formats briefly described as bead-based ELISAs. This high-throughput assay format works without any wash steps making this a fast and reproducible method.
Alpha technology relies on two antibodies that bind to the protein of interest at different epitopes. In the example shown above, one antibody binds to LRRK2 kinase; the second antibody binds to the phospho-site of the kinase to detect autophosphorylation. Acceptor and donor beads, each covered with one antibody, are brought together when the antibodies bind the same protein. Laser excitation of the donor beads releases thousands of singlet oxygens which can travel up to 200 nm in solution allowing the investigation of larger protein complexes (in comparison, the reach of TR-FRET is only 10 nm). The singlet oxygen initiates a cascade of reactions inside the acceptor beads resulting in luminescence/fluorescence.