BACH2 Antibody (for Western Blot) from Origene

June 13, 2016

Immunology
Duke University
Graduate student

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Origene

Product Name:

Rabbit Polyclonal Anti-BACH2 Antibody

Catalog Number:

TA331784

We needed to validate a retroviral expression vector so we constructed to confirm that it could express mouse Bach2 (transcript variant 1) protein in cells. This antibody was chosen because it is guaranteed to work with western blotting, and the vendor's product page indicates that it reacts with mouse Bach2 (although the immunogen is a peptide derived from human Bach2).

Experimental Design and Results Summary

Applications

Western Blot

Sample

Reduced and denatured whole-cell lysate from FACS-sorted 293T cells (GFP+) transfected with expression vector encoding mouse Bach2 (isoform 1) and GFP

Primary Incubation

1:2500 dilution (in PBS + 1% casein), 60 min, rocking, room-temperature

Blocking Agent

Block: PBS + 1% casein; Wash: PBS + 0.1% Tween-20

Secondary Incubation

Goat anti-Rabbit IgG (H+L), Alexa Fluor® 680 conjugate (1:5000 in PBS + 1% casein), 60 min, rocking, room-temperature

Tertiary Incubation

N/A

Detection

Fluorescence detection on a LI-COR digital imaging station

Results Summary

The antibody can successfully detect both human and mouse Bach2 in western blotting applications. Minor non-specific staining at ~50 kDa and ~30 kDa was detected, even in cells transfected with empty control vector. The apparent MW of human and mouse Bach2 (~104 kDa) is substantially higher than the predicted 92 kDa; however, we confirmed this is the correct band by running recombinant human Bach2 (Origene #TP314061) under identical denaturing SDS-PAGE conditions, then staining the gel with Coomassie Blue -- this protein was also ~105 kDa.

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Cross-reactive with mouse Bach2, reasonably good sensitivity and specificity

The Bad

Minor non-specific binding; not validated by vendor for ELISA, Flow cytometry, or immunoprecipitation

The Bottom Line

This is a reasonably good antibody for western blotting. We will continue to use it throughout the course of the project, and will order more of the same if we run out.

Share your experience with other scientists. Write a Review! »

Join the discussion