PathHunter™ EAstern Blot Assay Kits

 

The PathHunter™ EAstern Blot Assay Kit offers a new antibody-free method for recombinant protein characterization based on β-galactosidase complementation
that is exceptionally fast and simple. It is a convenient alternative to Western blots for monitoring recombinant protein size and relative abundance in cells expressing ProLabel (PL) or ProLink (PK)-tagged proteins. The kit may also be used to characterize these proteins in PathHunter applications. Researchers can generate double-stable cell lines by transfecting PathHunter Parental Cell Lines with ProLabel/ProLink cloning or expression vectors.

Product Features

•Exceptionally fast and simple protocol with no wash or blocking steps
•Antibody-free method reduces non-specific binding
•Compatible with many PathHunter applications

How does the EAstern Blot works

The EAstern Blot Assay Kit detects PL or PK-tagged recombinant proteins using
EFC complementation. After protein transfer, the EAstern blot process takes only
1.5 hours compared to approximately 4 hours for a Western blot. Blocking and wash steps are unnecessary, substantially streamlining the protocol. With no antibody present, non-specific binding does not occur, delivering exceptionally clean background.

The simple procedure involves transferring total cellular protein to PVDF or nitrocellulose membrane, rinsing the blot briefly with water and adding EA
Reagent. The PL/PK tag fused to the recombinant protein is a small fragment of
β-galactosidase that complements with the larger EA fragment on the membrane surface, reconstituting active enzyme. After 1 hour, Substrate Working Reagent is added and the active β-galactosidase hydrolyzes substrate on the membrane, releasing a chemiluminescent signal proportional to the amount of tagged protein present.

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(click on figure to enlarge)
Figure 1. Comparison of EAstern and Western Blots
Varying amounts of cell lysate (µg total protein) from CHO cells stably expressing NFAT-PL protein were separated by SDS-PAGE and transferred to nitrocellulose. The blot was divided in half and analyzed either by EAstern or western blot. The western blot utilized an anti-NFAT monoclonal antibody followed by an HRP-conjugated secondary antibody and chemiluminescent substrate. Comparable levels of sensitivities were observed for the two technologies, however a higher background was associated with the western blot.

(click on figure to enlarge)
Figure 2. EAstern Detection of ProLabel (PL)-tagged Protein
Cell lysate from CHO cells stably expressing NFAT-PL or transiently expressing cJun-PL or cyclin
D-PL (0.5-0.05 µg total protein) were separated by SDS-PAGE and transferred to nitrocellulose. The major bands representing full length tagged protein and minor bands representing degradation products were observed. No signal was detected from control CHO-KI cell lysates isolated from a mock transfection.

Available Collaterals

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