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| Product Name | His-Tag ELISA Detection Kit, Colorimetric, Anti-6xHis Monoclonal Antibody |
| Catalog No. | PTR-HMM-0092 |
| Description | Sandwich ELISA kit for quantitative detection and quantitation of recombinant proteins expressing polyhistidine (6xHis, His6) affinity tags. The assay uses a pre-coated 96-well microplate with an anti-6xHis tag monoclonal capture antibody adsorbed to the surface. His-tagged proteins in the sample bind specifically to the capture antibody during a 1-2 hour incubation. After washing, a biotinylated anti-6xHis detection antibody (recognizing a different epitope of the His-tag or the tagged protein backbone) is added, followed by streptavidin-horseradish peroxidase (HRP) conjugate and TMB (3,3',5,5'-tetramethylbenzidine) substrate. The resulting yellow color (absorbance at 450 nm) is directly proportional to the concentration of His-tagged protein. The kit includes a His-tagged protein standard (typically GFP-His6 or similar) for generating a standard curve, enabling absolute quantification of target proteins without requiring purified protein standards. |
| Intended Use | Quantitative detection of His-tagged recombinant proteins in: E. coli, yeast, insect cell (baculovirus), and mammalian cell (HEK293, CHO) expression lysates; cell culture supernatants (for secreted His-tagged proteins); purification fractions (IMAC eluates, column chromatography fractions); in-process quality control for recombinant protein production; and expression level comparison across constructs and conditions. |
| Principle / Technology | Anti-6xHis monoclonal capture antibody (adsorbed on microplate) binds His-tagged protein via the polyhistidine tag (Kd approximately 10^-8 to 10^-9 M for 6xHis). After washing away unbound proteins, a biotinylated anti-6xHis detection antibody binds to a second epitope on the His-tag, forming a sandwich. Streptavidin-HRP binds to the biotinylated detection antibody via the biotin-streptavidin interaction. TMB substrate is converted by HRP to a blue product (A650), which becomes yellow (A450) upon addition of stop solution (sulfuric acid). Signal intensity is proportional to His-tagged protein concentration. |
| Detection Method | 1) Add 100 uL His-tagged protein standard or sample (diluted in sample diluent) to wells; incubate 1-2 h at RT or overnight at 2-8 C with shaking; 2) Wash 4x with 300 uL Wash Buffer; 3) Add 100 uL Biotinylated Detection Antibody; incubate 1 h at RT; 4) Wash 4x; 5) Add 100 uL Streptavidin-HRP Conjugate; incubate 30 min at RT; 6) Wash 4x; 7) Add 100 uL TMB Substrate; incubate 15-30 min at RT in dark; 8) Add 100 uL Stop Solution; 9) Read A450 within 30 min; 10) Calculate concentration from standard curve (4-parameter logistic fit). |
| Sample Type | E. coli lysates (soluble and insoluble fractions after denaturant removal), yeast lysates, baculovirus-infected insect cell lysates, mammalian cell lysates, cell culture supernatants, IMAC column eluates. Sample preparation: centrifuge lysates 10,000 x g, 10 min, 4 C to remove debris before assay. |
| Performance Range / Specifications | Detection range: 0.1-100 ng/mL His-tagged protein (depending on protein size and tag accessibility); standard curve range: 0.16-10 ng/mL (typical 7-point standard curve); assay duration: 3.5-5 hours (or overnight + 3 h); sample volume: 100 uL per well. |
| Sensitivity / LOD | Detection limit: typical 0.1 ng/mL His-tagged GFP (approximately 3.5 pM for 27 kDa protein); approximately 10-fold more sensitive than SDS-PAGE with Coomassie staining for comparable proteins. |
| Specificity | Anti-6xHis monoclonal antibodies specifically recognize the 6xHis peptide sequence; cross-reactivity with endogenous histidine-rich mammalian proteins: background signal typically <0.1 ng/mL in untransfected cell lysates; minimal cross-reactivity with 10xHis, FLAG, HA, c-Myc, GST, or MBP tags. |
| Reaction Conditions / Protocol | Capture incubation: 1-2 h RT (or overnight 2-8 C); detection antibody incubation: 1 h RT; streptavidin-HRP: 30 min RT; TMB development: 15-30 min RT dark; all incubations with gentle shaking (300 rpm orbital shaker recommended). |
| Components / Formulation | Anti-6xHis Antibody Coated 96-Well Microplate (12 x 8-well strips in frame), His-Tagged Protein Standard (lyophilized, GFP-His6 or similar, 1 vial), Biotinylated Anti-6xHis Detection Antibody (100x concentrate, 120 uL), Streptavidin-HRP Conjugate (100x concentrate, 120 uL), Sample Diluent (25 mL), Wash Buffer (20x concentrate, 50 mL), TMB Substrate Solution (12 mL), Stop Solution (0.5 M H2SO4, 12 mL), Plate Sealers (4 sheets), Protocol. |
| Storage Conditions | Microplate, Sample Diluent, Wash Buffer, TMB, and Stop Solution at 2-8 C; Standard, Detection Antibody, and Streptavidin-HRP at -20 C; protect TMB and Streptavidin-HRP from light. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (96 determinations — one 96-well plate). |
| Product Form | Pre-coated 96-well strip plate; liquid reagents; lyophilized standard. |
| Quality Control | Each lot: standard curve R2 >0.99 (4-PL fit); detection limit <0.1 ng/mL (mean blank + 3 SD); spike recovery: 85-115% for 0.5-5 ng/mL His-GFP in E. coli lysate matrix; intra-assay CV <8%; inter-assay CV <12%; plate uniformity: <10% CV across plate. |
| Key Features | Pre-coated anti-6xHis plate (ready-to-use); biotin-streptavidin signal amplification; TMB colorimetric detection; includes His-tagged protein standard; sensitive to 0.1 ng/mL; compatible with common expression systems; strip plate format for partial use. |
| Purity | Anti-6xHis monoclonal antibody: >95% pure by SDS-PAGE; His-tagged standard: >95% pure by SDS-PAGE; all reagents: analytical grade or higher. |
| Concentration | His-tagged standard: reconstituted at 100 ng/mL (prepare serial dilution for standard curve: 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0 ng/mL); Detection Antibody: 100x concentrate (working dilution 1:100); Streptavidin-HRP: 100x concentrate (working 1:100). |
| Activity / Unit Definition | Streptavidin-HRP specific activity: >200 U/mg (1 U = amount producing 1 mg purpurogallin in 20 s at pH 6.0, 20 C from pyrogallol); anti-6xHis antibody affinity: Kd ~10^-9 M for 6xHis peptide. |
| Molecular Weight | His-tagged GFP standard: approximately 27 kDa (GFP ~27 kDa plus His-tag ~1 kDa); Antibody: IgG approximately 150 kDa; Streptavidin-HRP: approximately 90-120 kDa (streptavidin ~60 kDa + HRP ~44 kDa). |
| Source / Origin | Anti-6xHis antibodies: mouse monoclonal (hybridoma-derived, purified by Protein A/G affinity); streptavidin: recombinant from Streptomyces avidinii expressed in E. coli; HRP: purified from horseradish; His-tagged standard: recombinant expressed in E. coli; all other reagents: synthetic or recombinant. |
| pH Range / Optimal pH | Sample Diluent pH 7.2-7.4; Wash Buffer pH 7.4; TMB substrate pH ~4.0; Stop Solution pH <1.0; optimal binding pH 7.0-8.0. |
| Shipping Conditions | Cold pack (2-8 C) for most components; Standard, Detection Antibody, and Streptavidin-HRP may ship on dry ice. |
| Expiration Date / Stability | 12 months at recommended conditions; reconstituted standard stable at 2-8 C for 1 week or at -20 C for 3 months; plate strips stable in sealed pouch with desiccant after opening for 4 weeks at 2-8 C. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. Kit contains components of animal origin (mouse antibodies, bovine-derived reagents in culture medium) — country-specific import may apply. |
| Compatibility | Compatible with His-tagged proteins from E. coli (soluble and denaturant-removed inclusion body preparations), yeast (S. cerevisiae, P. pastoris), insect cells (Sf9, Sf21, High Five), and mammalian cells (HEK293, CHO). Denaturants: urea up to 0.5 M, guanidine-HCl up to 0.2 M tolerated in final sample dilution (higher concentrations may affect antibody binding). Imidazole: up to 10 mM in final sample tolerated (higher imidazole concentrations from IMAC elution buffer must be diluted, dialyzed, or desalted). Reducing agents (DTT up to 1 mM, beta-mercaptoethanol up to 10 mM) do not interfere. EDTA up to 5 mM tolerated. The 6xHis tag must be accessible (N-terminal, C-terminal, or surface-exposed internal loop) — buried tags in aggregated or misfolded proteins may not be detected. |
| Recommended Buffer System | Sample Diluent: PBS pH 7.4, 1% BSA, 0.05% Tween-20, 0.05% ProClin 300; Wash Buffer: PBS pH 7.4, 0.05% Tween-20; TMB Substrate: TMB, H2O2, citrate-phosphate buffer pH ~4.0. |
| Application Notes / Precautions | Reconstitute His-tagged protein standard in Sample Diluent or PBS as specified on vial; aliquot and store at -80 C to avoid repeated freeze-thaw. For unknown samples, test multiple dilutions (undiluted, 1:10, 1:100, 1:1,000) in Sample Diluent to ensure readings fall within the standard curve. Cell lysates and column eluates should be centrifuged (10,000 x g, 5-10 min) before assay to remove particulates. If imidazole concentration in sample is >10 mM, desalt or dialyze against PBS before assay. Incubate plate with shaking for optimal sensitivity. For overnight capture incubation, seal the plate to prevent evaporation. TMB substrate incubation: obtain optimal signal at 15-30 min; if blue color develops very rapidly (<5 min), dilute sample further and repeat. |
| Batch-to-Batch Consistency | Standard curve R2 >0.99 for every lot; LOD <0.1 ng/mL; spike recovery 85-115%; intra-assay CV <8%. |
For research use only, not for clinical use.
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