HiBiTタグとシンプルウェスタンによる、高精度なタンパク質定量

Application Note Simple Western HiBiT Assay Highly Specific Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology Automated Analysis of HiBiT-Protein Expression, Isoforms, Targeted Protein Degradation, and More Introduction Accurate quantification of tagged proteins plays a pivotal determination to characterize hits identified via role in a broad array of biological applications, including HiBiT plate screens. protein expression profiling, isoform characterization, assay validation, and targeted protein degradation (TPD) Key Takeaways: studies. The HiBiT protein tagging system, developed by • High-throughput, high-specificity antibody-based Promega, provides a sensitive and versatile method for detection of HiBiT-tagged proteins, offering a powerful protein detection through an 11–amino acid peptide tag. orthogonal approach to plate-based luminescent The HiBiT tag binds to its complementary subunit LgBiT HiBiT assays. to reconstitute NanoBiT® luciferase, a bright, luminescent enzyme, enabling rapid protein quantification in multiple • Capable of processing up to 96 samples in ~3 hours, formats, including lytic and live-cell plate-based assays. combining throughput with molecular weight resolution, A high-affinity anti-HiBiT monoclonal antibody expands ideal for efficient screening follow-up or standalone the utility of the HiBiT-tag, enabling traditional epitope tag protein analysis analysis of HiBiT-tagged proteins. • Demonstrated sensitivity exceeding traditional Western While plate-based HiBiT assays are fast and high- blot and broad dynamic and linear ranges throughput, they lack the molecular-weight resolution required to verify tagged-protein identity and to • Quantitative DC₅₀ and Dmax characterization of distinguish isoforms or degradation products. degradation hits identified in HiBiT screens, providing Simple Western™ Technology addresses this gap with enhanced resolution and isoform specificity automated capillary-based immunoassays that integrate HiBiT CP Total Protein protein separation, immunoprobing, and detection on a 16 30 single platform. Compared with traditional western blots, 14 25 Simple Western offers markedly higher sensitivity along 12 with excellent reproducibility, total-protein normalization, 20 10 and multiplexing capabilities, making it an effective 8 15 orthogonal complement to plate-based assays. 6 10 In this application note, we evaluate the performance of 4 the Anti-HiBiT Monoclonal Antibody from Promega using 5 2 Simple Western Technology (Fig. 1). Simple Western assays that utilize Anti-HiBiT Monoclonal Antibody 0 0 12 40 66 116 180 230 provide an advanced solution for validating hits from MW (kDa) high-throughput plate-based HiBiT screens. This study Figure 1. Detection of HiBiT Control Protein (CP) in human lysate using provides protocol guidance for the Simple Western HiBiT the Anti-HiBiT Monoclonal Antibody (Promega) with Simple Western mAb Assay and demonstrates the use of the Technology (Bio-Techne). Total protein (blue trace) was detected in the Simple Western for downstream DC₅₀ and Dmax same capillary using the RePlex Module. HiBiT CP Chemiluminescence (RLU × 10,000) Total Protein Chemiluminescence (RLU × 100)

Materials and Methods To optimize antibody performance, HiBiT Control Protein Establishing the Simple Western HiBiT Assay (CP) was diluted to 10 ng/mL in 0.1 mg/mL human protein extract to simulate complex biological samples. Samples HiBiT detection was performed using the Anti-HiBiT were heat-denatured under reducing conditions and Monoclonal Antibody (clone 30E5). Simple Western analyzed on both Leo and Jess. For Leo, the Anti-HiBiT analysis was performed using Leo™ and Jess™ Systems. Monoclonal Antibody was tested in a 12-point, 2-fold Additional consumables and reagents for protein analysis titration series ranging from 100 – 0.049 µg/mL with four were provided by Promega (Table 1) and Bio-Techne replicates. Jess experiments used the antibody in an (Table 2). 8-point, 3-fold titration series from 100 – 0.046 µg/mL with three replicates. HiBiT signal was normalized to total TA B L E 1 . protein measured in the same capillary using the Promega materials used in this study. RePlex Module. Name Cat. # To define assay sensitivity, range, standard curve, and recovery, HiBiT CP was prepared in a 10-point, 5-fold Anti-HiBiT Monoclonal Antibody N7200  dilution series from 5 µg/mL down to 2.56 pg/mL in HiBiT Control Protein N3010 0.1 mg/mL human protein extract. The same batch of Mass Spec-Compatible Human sample was prepared for analysis on Leo and Protein Extract V6941 Jess Systems. Anti-HiBiT Monoclonal Antibody Anti-Mouse IgG (H+L), HRP Conjugate W4021 concentration was fixed at 10 µg/mL, the saturation point determined in earlier optimization. ECL Western Blotting Substrate W1015 Comparison to Traditional Western Blot TA B L E 2 . For comparison to traditional Western blot, the HiBiT CP Bio-Techne materials used in this study. was prepared in a 7-point, 2-fold titration ranging from 2 – 0.031 ng/mL in 0.1 mg/mL human protein extract in triplicate. The same batch of sample was split and loaded Name Cat. # in equivalent volumes for analysis by Western blot and Simple Western. For detection by Western blot, Anti- 12-230 kDa Separation Module (Jess) SM-W001 HiBiT Monoclonal Antibody was diluted to 1 µg/mL and 12-230 kDa Separation Module (Leo) SWSM-W014 incubated overnight at 4 °C with gentle shaking. Anti- Mouse IgG (H+L), HRP Conjugate was diluted to 0.2 µg/ EZ Standard Pack 1 PS-ST01EZ-8 mL and incubated 1 hour at room temperature with gentle Anti-Mouse Detection Module DM-002 shaking. ECL Western Blotting Substrate was added and Anti-Rabbit Detection Module DM-001 then the membrane was imaged on a FluorChem R Imager. Detection with Simple Western was performed using Total Protein Detection Module SWDM-TP21 the Anti-Mouse Detection Module. Simple Western and RePlex™ Module RP-001 traditional Western blot exposures were 128 seconds for Anti-Mouse HRP Secondary direct comparison. Antibody 100X 040-655 Anti-BRD4 rabbit pAb (C-terminus) NBP1-86640 Anti-BRD4 rabbit mAb (N-terminus) NBP3-15452 Anti-GAPDH mouse mAb MAB5718 High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 02

BRD4 Targeted Protein Degradation BRD4, and C-terminal BRD4, respectively. Protein A549 cells edited via CRISPR/Cas9 to insert the HiBiT tag expression measurements were normalized to GAPDH at the N-terminus of the BRD4 gene were treated for detected in the same capillary using the RePlex Module 6 hours with vehicle only or treated with Compound 8, using mouse anti-GAPDH antibody prepared at a BRD4-targeting PROTAC® Degrader, at concentrations 50 µg/mL concentration. Due to high (2 mg/mL) lysate (nM) of 0.1, 0.5, 2, 4, 8, 32, and 125. Samples were concentration, Anti-Mouse HRP Secondary Antibody 100X analyzed at concentrations (mg/mL) of 2.0, 0.5, and diluted to 1X was used for HiBiT detection, while the 0.1 for detection using antibodies targeting HiBiT, Anti-Mouse HRP Detection Module was used for BRD4 N-terminal BRD4, and C-terminal BRD4, respectively. (N- and C-term) and GAPDH detection. DC50 and Dmax Primary antibodies were prepared at concentrations values were calculated using GraphPad Prism with a (µg/mL) of 10, 40, and 20 targeting HiBiT, N-terminal 4-parameter logistic (4-PL), unweighted curve fit. Results Defining the Simple Western HiBiT Assay Optimization experiments demonstrated that the Anti-HiBiT Monoclonal Antibody reached saturation at 10 µg/mL on both Leo and Jess platforms (Fig. 2). Signal intensity increased with antibody concentration before plateauing, indicating effective saturation for subsequent quantitative experiments. A r Leo er Jess adde Anti-HiBiT mAb add Anti-HiBiT mAb kDa L L kDa 230 230 180 180 116 116 66 66 40 HiBiT CP 40 HiBiT CP (Probe 1) (Probe 1) 12 12 230 230 180 180 116 Total Protein 116 Total Protein (Probe 2) (Probe 2) 66 66 40 40 12 12 Figure 2: Optimization of HiBit CP Detection using the Anti-HiBit Monoclonal Antibody on Leo (Left) and Jess (Right). Lane view High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 03

B 5 Raw Area 20 Raw Area Area Normalized to Total Protein Area Normalized to Total Protein 4 15 3 10 2 5 1 0 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 Anti-HiBiT mAb (μg/mL) Anti-HiBiT mAb (μg/mL) Figure 2. Optimization of HiBiT CP detection using the Anti-HiBiT Monoclonal Antibody on Leo (left) and Jess (right). (A) Lane view of HiBiT detection using the Anti-HiBiT Monoclonal Antibody and Total Protein Detection performed during the same run using the RePlex feature on Leo. (B) Antibody saturation curves of average peak areas resulting from HiBiT detection using the Anti-HiBiT Monoclonal Antibody, including raw values (black trace) and values normalized to total protein, shown in the blue trace and orange traces for Leo and Jess, respectively. Error bars represent standard deviations from the means. High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 04 HiBiT Peak Area × 10,000 HiBiT Peak Area × 10,000

The Simple Western HiBiT assay achieved a dynamic range exceeding four orders of magnitude (0.32 – 5000 ng/mL), with a linear range of approximately 2.5 logs (0.32 – 200 ng/mL) (Fig. 3). This broad quantitative range allows for flexible assay design across varied expression levels. Dynamic Range Leo Jess 9 14 8 12 7 10 6 5 8 4 6 3 4 2 1 2 0 0 0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000 HiBiT CP (ng/mL) HiBiT CP (ng/mL) Linear Range Leo Jess 14 35 12 30 10 R2 = 0.9993 25 R2 = 0.9938 8 20 6 15 4 10 2 5 0 0 0 50 100 150 200 0 50 100 150 200 HiBiT CP (ng/mL) HiBiT CP (ng/mL) Figure 3. Dynamic and linear ranges of HiBiT detection using the Anti-HiBiT Monoclonal Antibody on Leo and Jess. High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 05 Area x 100,000 Area x 1,000,000 Area x 100,000 Area x 1,000,000

Comparisons with traditional Western blot showed that both Leo and Jess were significantly more sensitive, detecting HiBiT CP at concentrations as low as 0.1 ng/mL, compared to 0.5 ng/mL for traditional blotting (Fig. 4). Limit of detection (LOD) values were 0.11 ng/mL for Jess and 0.24 ng/mL for Leo, compared to 0.66 ng/mL for Western blot (Table 3). Similarly, limit of quantification (LOQ) values further underscored the improved sensitivity of Simple Western over conventional methods (Table 3). A Leo Jess B HiBiT CP HiBiT CP (ng/mL) 7 HiBiT CP HiBiT CP (ng/mL) kDa 5 1.00 6 0.25 250 0.50 0.13 0.25 0.06 150 4 0.13 5 0.03 0.00 0.00 100 75 3 4 2 3 50 2 37 1 25 1 20 0 0 15 10 12 40 66 116 180230 12 40 66 116 180230 2.0 1.0 0.5 0.3 HiBiT CP MW (kDa) MW (kDa) (ng/mL) Figure 4. Sensitivity of HiBiT CP detection using the Anti-HiBiT Monoclonal Antibody by Simple Western Leo and Jess and traditional Western blot. (A) Electropherograms of HiBiT CP Detection using Leo and Jess (B) traditional Western blot detection of HiBiT CP. The exposure for both Simple Western and traditional Western blot was 128 seconds. Parameter Leo Jess Western blot LOD ng/mL (pg/well) 0.24 (0.72) 0.11 (0.33) 0.66 (1.98) LOQ ng/mL (pg/well) 0.71 (2.13) 0.34 (1.02) 1.62 (4.86) Table 3. LOD and LOQ of the HiBiT detection using the Anti-HiBiT Monoclonal Antibody on Leo and Jess and comparison to traditional Western blot. High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 06 Chemiluminescence (RLU × 100) Chemiluminescence (RLU × 100)

Standard curve analysis demonstrated strong assay reproducibility, with log-log nonlinear regression (1/y2 weighting) yielding R2 values greater than 0.99 and recovery rates near 100% (Fig. 5). These data confirm that Simple Western can reliably and quantitatively detect HiBiT-tagged proteins with high precision. Standard Curve Leo Jess 107 107 R2 = 0.9920 R2 = 0.9961 106 106 105 105 104 104 103 103 0.1 1 10 100 1000 0.1 1 10 100 1000 HiBiT CP (ng/mL) HiBiT CP (ng/mL) % Recovery Leo Jess 150 150 100 100 50 50 0 0 0.1 1 10 100 1000 0.1 1 10 100 1000 HiBiT CP (ng/mL) HiBiT CP (ng/mL) Figure 5. Standard curve and recovery of HiBiT CP detection using the Anti-HiBiT Monoclonal Antibody on Leo and Jess. High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 07 % Recovery Total Protein Normalized Area % Recovery Total Protein Normalized Area

Utility of the Simple Western HiBiT Assay in Targeted Protein Degradation To illustrate the assay’s relevance in targeted protein degradation workflows, we applied the method to monitor BRD4 degradation, a transcriptional co-activator implicated in cancer, in response to Compound 8, a next-generation PROTAC® Degrader.1 A similar dose-dependent decrease in HiBiT-BRD4 signal in a breast cancer cell line (A549) was observed with all three antibodies targeting HiBiT, N-terminal BRD4, and C-terminal BRD4 (Fig. 6). Importantly, Simple Western separates full-length from truncated BRD4 isoforms, adding molecular-weight context that complements plate-based luminescent readouts. Leo er d Compound 8 La d kDa 230 BRD4 Long Isoform 180 BRD4 Short Isoforms 116 66 40 12 Anti-HiBiT Anti-BRD4 (N-term) Anti-BRD4 (C-term) Jess er dd Compound 8 La kDa 230 BRD4 Long Isoform 180 BRD4 Short Isoforms 116 66 40 12 Anti-HiBiT Anti-BRD4 (N-term) Anti-BRD4 (C-term) Figure 6. BRD4 degradation by dosing with Compound 8 detected using the Anti-HiBiT Monoclonal Antibody and antibodies targeting N-terminal BRD4 and C-terminal BRD4. Cells were treated with Compound 8, a BRD4-targeting degrader, at concentrations of 0.1, 0.5, 2, 4, 8, 32, and 125 nM (shown left to right). High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 08

Dose-response curves derived from long BRD4 isoform Anti-HiBiT signals normalized to the vehicle-only control produced nearly identical trends across detection strategies Anti-BRD4 (N-term) 100 (Fig. 7). Consistent DC Anti-BRD4 (C-term) 50 and Dmax values were obtained, validating the Anti-HiBiT Monoclonal Antibody detection method for characterizing BRD4 degradation (Fig. 8). Together, these results demonstrate that HiBiT tagging provides results consistent with traditional BRD4 antibody 50 detection, while Simple Western adds the ability to resolve isoform-specific responses, offering deeper insight into degrader-driven protein dynamics. 0 Ctrl 0.1 1 10 100 1000 Compound 8 (nM) Figure 7. Dose-response analysis of BRD4 degradation by Compound 8 detected using 3 antibodies targeting HiBiT, N-terminal BRD4, and C-terminal BRD4 normalized to the vehicle-only control. Leo Jess 3.0 Raw DC50 120 Raw Dmax 2.60 2.43 100 96.1 95.6 95.5 96.5 98.1 95.6 2.0 80 1.57 1.59 1.39 1.41 60 1.0 40 20 0 0 HiBiT BRD4 (N-term) BRD4 (C-term) HiBiT BRD4 (N-term) BRD4 (C-term) Antibody Target Antibody Target Normalized DC50 120 Normalized Dmax 6 1.53 1.60 1. 96.2 96.3 94.8 96 97.8 100 .4 94.9 1.2 80 0.97 0.86 60 0.8 0.61 40 0.39 0.4 20 0 0 HiBiT BRD4 (N-term) BRD4 (C-term) HiBiT BRD4 (N-term) BRD4 (C-term) Antibody Target Antibody Target Figure 8. DC50 and Dmax values calculated from BRD4 degradation by Compound 8 detected using 3 antibodies targeting HiBiT, N-terminal BRD4, and C-terminal BRD4 on Leo (blue bars) and Jess (orange bars). Values were normalized to GAPDH. High-Resolution Protein Quantification Using an Anti-HiBiT Monoclonal Antibody and Simple Western Technology 09 Compound 8 DC50 (nM) Compound 8 DC50 (nM) Dmax (%) Dmax (%) % of Vehicle-only Ctrl

Conclusion R E F E R E N C E S 1. Actis M, Cresser-Brown J, Caine EA, et al. Evaluation of Cereblon- Simple Western Technology provides a robust, highly Directing Warheads for the Development of Orally Bioavailable specific platform for the quantitative detection of PROTACs. J Med Chem. 2025 Feb 13;68(3):3591-3611. HiBiT-tagged proteins using the Anti-HiBiT Monoclonal 2. Prindle V, Richardson AE, Sher KR, et al. Synthetic lethality Antibody from Promega. Simple Western provides of mRNA quality control complexes in cancer. Nature. 2025 molecular weight resolution to determine molecular Feb;638(8052):1095-1103. weight variants and protein isoforms, complementing PROTAC® is a registered trademark of Arvinas Operations, Inc., and is plate-based assays. Furthermore, Simple Western used under license. provides superior sensitivity, reproducibility, and automation compared to traditional Western blot methods, supporting a wide dynamic range and enables sub-picogram protein quantification. Its utility extends from protein expression and isoform analysis to TPD workflows, where accurate monitoring of degradation kinetics is essential. The Anti-HiBiT Monoclonal Antibody may also be used with Simple Western in co-immunoprecipitation studies for identifying protein- protein interactions.2 When paired with the Anti-HiBiT Monoclonal Antibody, Simple Western becomes a valuable orthogonal tool for confident characterization of tagged proteins in both basic and translational research, offering a unified workflow where Simple Western is directly integrated into target screening using Promega’s Anti-HiBiT Monoclonal Antibody, yielding higher specificity with up to 96 samples analyzed in just 3 hours. For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners. 18005539605_0925 Global Developer, Manufacturer, and Supplier of High-Quality Reagents, Analytical Instruments, and Precision Diagnostics. INCLUDES R&D Systems™ Novus Biologicals™ Tocris Bioscience™ ProteinSimple™ Asuragen® ACD™ Lunaphore™