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Mouse Anti-HiBiT mAb (Clone 30E5)

Sensitive Antibody-Based Detection of HiBiT-Tagged Proteins

  • Confirm subcellular localization of endogenous HiBiT-tagged proteins in cell pools or clones following CRISPR/Cas9 HiBiT knock-in
  • Use as an orthogonal method to verify bioluminescent results
  • Confirm protein levels and size using classic Western blot detection
  • Perform immunoprecipitation of HiBiT-tagged proteins
  • Perform FACS analysis on live cells (extracellular HiBiT) or on fixed cells (intracellular HiBiT)
  • Early access material; please contact us for more information
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Mouse Anti-HiBiT mAb (Clone 30E5)

Immunodetection of HiBiT-Tagged Proteins

The 11-amino-acid HiBiT peptide tag can be added to a protein of interest using traditional cloning or CRISPR/Cas9 genome editing, enabling analysis of proteins under endogenous expression conditions. HiBiT-tagged proteins are typically measured using the bioluminescent signal generated upon addition of Nano-Glo® HiBiT detection reagents, providing for precise measurement of changing protein levels over 7 logs of linear dynamic range.

Anti-HiBiT mAb is a specific and highly sensitive monoclonal antibody (mAb) that expands the detection options for HiBiT-tagged proteins, enabling traditional antibody-based methods for detection of the HiBiT tag.

Sensitivity and Specificity of Anti-HiBiT mAb is Comparable to or Better than Common Epitope Tag Antibodies

Western blot detection of HiBiT-tagged proteins demonstrates specific detection of HiBiT-tagged proteins with signal-to-background ratio similar to or better than a single copy of FLAG®, Myc or HA tags when detected with commonly used antibodies.

The images to the right compare Western blot detection using anti-HiBiT mAb and common epitope tag antibodies. HaloTag-HiBiT and a multi-tag protein (Genscript Multiple Tag) that contains a single copy of FLAG®, Myc and HA tags were serially diluted into HEK293 lysate so that 0, 10pg, 100pg, 1ng or 10ng of the tagged protein was loaded in each lane. After SDS-PAGE and transfer to PVDF membrane, Western blotting was performed with 1µg/ml primary antibody, 0.4µg/ml Anti-Mouse IgG (H+L), HRP Conjugate (Promega), or anti-rat (R&D Systems, anti-HA blot) secondary antibody and ECL Western Blotting Substrate (Promega) using a 144s exposure.

Comparison of Western blot detection using anti-HiBiT mAb and common epitope tag antibodies.

Subcellular Localization Following CRISPR/Cas9 HiBiT Knock-In

Immunofluorescence staining using the anti-HiBiT mAb allows characterization of HiBiT knock-in cell pools and clones to confirm that the endogenously expressed HiBiT-tagged protein has the expected subcellular localization.

Detection of HiBiT-tagged proteins in CRISPR-edited cell pools and clones using the Anti-HiBiT mAb.

Detection of HiBiT-tagged proteins in CRISPR-edited cell pools and clones using the Anti-HiBiT mAb. CRISPR/Cas9 editing was used to knock in HiBiT at the endogenous locus of proteins with varying subcellular localization in HeLa cells. CRISPR-modified clones or pools of cells were imaged by epifluorescence on a Keyence BZ-X800 microscope with 40X or 60X magnification after staining with the Anti-HiBiT mAb and AlexaFluor® 647-labeled anti-mouse secondary antibody with Hoechst dye staining of the nucleus. Panel A. VCL-HiBiT pool at 40X magnification. Panel B. EEA1-HiBiT pool at 60X magnification. Panel C. HSP90B1-HiBiT pool at 60X magnification. Panel D. EGFR-HiBiT clone at 40X magnification.

Precipitation of HiBiT-Tagged Proteins from Cell Lysates

The anti-HiBiT mAb can be used to enrich HiBiT-tagged proteins for further analysis or to determine co-immunoprecipitation partners. This method provides improved immunoprecipitation efficiency compared to a single copy of FLAG® tag and similar efficiency to three copies of FLAG® tag sequence.

Comparison of FLAG-HiBiT and anti-HiBiT antibody in pull-down experiments.

Comparison of pull-down efficiency from CRISPR-modified cell pools. CRISPR/Cas9 gene editing was used to knock in either HiBiT, FLAG-HiBiT, HiBiT-FLAG, 3XFLAG-HiBiT or HiBiT-3XFLAG to the C-terminus of the endogenous locus of the HSP90B1 protein in HeLa cells. Lysates from pools of CRISPR-edited cells were diluted with buffer and divided 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 or M2 Anti-FLAG antibody as indicated. After a 2-hour incubation, resins were washed, and 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.

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