Fast Antibody-free Alternative to Western Blot
The PathHunter™ EAstern Blot Assay Kit offers a new antibody-free method for recombinant protein characterization based on ß-galactosidase complementation that is exceptionally fast and simple. After protein transfer, the EAstern process takes only 1.5 hours compared to about 4 hours for a Western blot. Blocking and wash steps are unnecessary, substantially streamlining the protocol, and with no antibody present, non-specific binding does not occur, delivering exceptionally clean background.
PathHunter Application Compatible
The EAstern Kit may be used to purify or characterize expressed ProLabel™-tagged protein in PathHunter applications. Researchers can generate double-stable cell lines by transfecting PathHunter EA-Nuc Cell Lines with ProLabel Cloning or Expression Vectors. Then for monitoring recombinant protein size and relative abundance, the EAstern Kit offers a fast and convenient alternative to Western Blots.
Features and Benefits
» Exceptionally fast & simple with no wash or blocking steps
» Antibody-free method reduces non-specific binding
» Compatible with many PathHunter applications
How the EAstern Blot Works
The EAstern Blot Assay Kit detects ProLabel-tagged recombinant proteins using EFC complementation. The simple procedure involves transferring total cellular protein to PVDF or nitrocellulose membrane, rinsing the blot briefly with water, and adding EA Reagent. The ProLabel tag fused to the recombinant protein is a small fragment of ß-galactosidase that complements with a larger EA fragment on the membrane surface, forming active enzyme. After 60 minutes, Substrate Working Reagent is added and the active ß-gal hydrolyzes substrate right on the membrane, releasing chemiluminescent signal proportional to the amount of ProLabel-tagged protein present.
» EAstern Blot Substrate
» Substrate Reconstitution Buffer
» Substrate Dilution Buffer
» EA Reagent
» Positive ProLabel-tagged Protein Control
» Luminometer, Multimode Microplate Reader or CCD Camera