Description
Anti-HiBiT Antibody Performs As Well As or Better Than Common Epitope Tag Antibodies
Western blot detection and immunoprecipitation of HiBiT-tagged proteins demonstrate specific detection and efficient pull-down of HiBiT-tagged proteins with performance similar to or better than FLAG®, Myc or HA tags when using common antibodies.
Comparison of Western blot detection using Anti-HiBiT Monoclonal Antibody to common epitope tag antibodies. HSP90B1 was endogenously tagged on the C-terminus in HeLa cells with HiBiT alone or HiBiT and a single copy of the Myc, HA or FLAG® tags, or three copies of the FLAG® tag. Lysates (10µg) from the HSP90B1-tagged cell pools were separated by SDS-PAGE. After transfer to PVDF membrane, Western blotting was performed with 1µg/ml Anti-HiBiT or Anti-Myc, 0.1µg/ml Anti-HA or 10µg/ml Anti-FLAG® primary antibodies (as indicated) and 0.4µg/ml anti-mouse or anti-rat (for anti-HA blot) secondary antibody and ECL substrate using a 5-minute exposure. The HiBiT antibody image brightness was scaled 0–29,817 and all other images 145–2735.
Comparison of pull-down efficiency from CRISPR-modified cell pools. CRISPR/Cas9 gene editing was used to knock in either HiBiT, HiBiT-FLAG, HiBiT-3XFLAG, HiBiT-HA or HiBiT-Myc to the C-terminus of the endogenous locus of the HSP90B1 protein. Lysates from pools of CRISPR-edited HeLa cells were diluted into buffer and split to generate equivalent samples for pull-down comparisons. The HSP90B1 protein was immunoprecipitated with magnetic Protein G resin (Cytiva) using 2µg of either the Anti-HiBiT, FLAG®, HA or Myc antibody as indicated. After a 2-hour incubation, resins were washed, and the proteins were eluted by heating in SDS loading buffer. The amount of tagged HSP90B1 protein precipitated from cell lysates was detected using the Nano-Glo® HiBiT Blotting System. Both eluted protein and the protein remaining in the supernatant are shown. The intensity of the signal in the starting sample compared to the antibody-treated supernatant indicates the protein clearance efficiency of the antibody.
