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| Product Name | Bradford Protein Assay Kit, Coomassie G-250, Detergent-Compatible, Colorimetric |
| Catalog No. | PTR-HMM-0090 |
| Description | Coomassie Brilliant Blue G-250 dye-binding protein assay kit for rapid colorimetric quantification of total protein in solution. Based on the Bradford method, the assay relies on the binding of Coomassie G-250 dye to basic and aromatic amino acid residues (primarily arginine, lysine, and histidine) in proteins under acidic conditions. Upon protein binding, the dye undergoes a spectral shift from its cationic reddish-brown form (Amax 465 nm) to the anionic blue form (Amax 595 nm), with the increase in absorbance at 595 nm directly proportional to protein concentration. The detergent-compatible formulation includes modifications to reduce interference from common laboratory detergents (up to 1% Triton X-100, 0.1% SDS, 1% NP-40), making it suitable for protein quantification in cell lysis buffers. The kit includes bovine serum albumin (BSA) and bovine gamma-globulin (BGG) protein standards for generating standard curves matched to the sample type. |
| Intended Use | Rapid total protein quantification in: cell lysates (RIPA, NP-40, Triton X-100-based); tissue homogenates; column chromatography fractions (affinity, ion exchange, size exclusion); purified and recombinant protein preparations; enzyme assay sample normalization; and protein concentration adjustment for SDS-PAGE and western blot loading. |
| Principle / Technology | Under acidic conditions (phosphoric acid, pH ~0.5), Coomassie G-250 exists in three forms: cationic (red, A465, unbound), neutral (green, A650), and anionic (blue, A595, protein-bound). Addition of protein stabilizes the anionic blue form through binding to arginine, lysine, histidine, and aromatic residues (primarily via electrostatic and van der Waals interactions). The dye-protein complex has a high extinction coefficient, providing sensitivity comparable to or exceeding the Lowry method. |
| Detection Method | Mix 5-50 uL protein sample with 200-250 uL Bradford Reagent in a 96-well plate or cuvette; incubate 5-10 min at RT; measure absorbance at 595 nm (or 570-620 nm); calculate protein concentration from BSA or BGG standard curve (0-2,000 ug/mL range); correction for sample buffer background: include buffer-only blank. |
| Sample Type | Cell lysates (RIPA, NP-40, Triton X-100, SDS-based), tissue homogenates, purified proteins, chromatography fractions, serum, plasma (diluted). Sample volume 5-50 uL; protein range 1-2,000 ug/mL. |
| Performance Range / Specifications | Detection range: 1-2,000 ug/mL BSA (standard microassay: 1-25 ug/mL; standard assay: 25-2,000 ug/mL); linear range: 25-1,500 ug/mL BSA (R2 >0.99); assay time: 5-10 min; color stability: 60 min at RT; detergent tolerance: 1% Triton X-100, 1% NP-40, 0.1% SDS, 1% CHAPS, 1% octyl glucoside. |
| Sensitivity / LOD | Detection limit: 1 ug/mL BSA (microassay protocol, 800 uL total volume); typical detection: 5-10 ug/mL in standard 96-well format (200 uL); 0.5-1 ug protein per well detectable. |
| Specificity | Coomassie G-250 binds primarily to arginine, lysine, and histidine residues; protein-to-protein variation in dye-binding results in different responses for different proteins (BSA vs BGG standard curves differ); the assay responds to peptides >3 kDa; non-protein sources of signal: strong bases, concentrated buffers, and some reducing agents; most non-interfering at standard concentrations. |
| Reaction Conditions / Protocol | Incubation: 5-10 min at RT; absorbance measured at 595 nm; color stable for 60 min (read within this window); reaction volume: 200-1,000 uL depending on cuvette or plate format. |
| Components / Formulation | Bradford Reagent (Coomassie G-250 in phosphoric acid and methanol, 500 mL), BSA Standard (2 mg/mL in 0.9% saline, 1 mL, 5 vials), BGG Standard (2 mg/mL in 0.9% saline, 1 mL), Protocol, 96-Well Clear Plate (2 plates). |
| Storage Conditions | Bradford Reagent at RT (protect from light — amber bottle); BSA and BGG standards at 2-8 C; standards at -20 C for long-term storage. |
| Shelf Life | Bradford Reagent: 24 months at RT; protein standards: 12 months at 2-8 C, 24 months at -20 C. |
| Package Specifications | 1 kit (500 mL reagent, sufficient for approximately 2,500 standard assays at 200 uL/assay, or 800 microassays at 1 mL/assay). |
| Product Form | Bradford Reagent: dark brown-red liquid; protein standards: clear colorless liquid. |
| Quality Control | Each lot tested with BSA standard curve (0-2,000 ug/mL): R2 >0.99, OD595 of 1,000 ug/mL BSA 0.4-0.6; reagent blank: A595 <0.3 against water; detergent compatibility verified (no precipitation with 1% Triton X-100); inter-assay CV <5% for BSA samples. |
| Key Features | Coomassie G-250 Bradford method; detergent-compatible (up to 1% Triton X-100, 0.1% SDS); rapid 5-10 min assay; single reagent; BSA and BGG standards included; color stable 60 min; 96-well plate and cuvette compatible. |
| Purity | Coomassie G-250: dye content >=80%; phosphoric acid: analytical grade; methanol: analytical grade; BSA standard: >98% pure by SDS-PAGE; BGG standard: >95% pure by SDS-PAGE. |
| Concentration | Bradford Reagent: ready-to-use; BSA standard: 2.0 mg/mL; BGG standard: 2.0 mg/mL; working range: 25-2,000 ug/mL standard assay, 1-25 ug/mL microassay. |
| Activity / Unit Definition | Not applicable — protein quantification assay. |
| Molecular Weight | Coomassie Brilliant Blue G-250: 854.02 g/mol; BSA: ~66.5 kDa; BGG: ~150 kDa (IgG). |
| Source / Origin | Coomassie G-250: synthetic dye; BSA: purified from bovine plasma (US origin, tested BSE-free); BGG: purified from bovine serum; all buffer components: analytical grade. |
| pH Range / Optimal pH | Bradford Reagent pH ~0.5 (acidic phosphoric acid); protein standard pH ~7.0 (in saline). |
| Shipping Conditions | Ambient temperature for Bradford Reagent; cold pack for protein standards. |
| Expiration Date / Stability | 24 months at RT for Bradford Reagent; 12 months at 2-8 C for protein standards; thawed BSA/BGG stable at 2-8 C for 4 weeks; avoid repeated freeze-thaw of standards. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. BSA and BGG standards are purified from bovine plasma — country-specific import regulations may apply. |
| Compatibility | Detergent compatibility: Triton X-100 (up to 1%), NP-40 (up to 1%), SDS (up to 0.1%), CHAPS (up to 1%), octyl-beta-glucoside (up to 1%), Tween-20 (up to 0.1%). Reducing agent tolerance: DTT (up to 1 mM), beta-mercaptoethanol (up to 10 mM), TCEP (up to 1 mM). Buffer tolerance: Tris (up to 100 mM), HEPES (up to 100 mM), phosphate (up to 50 mM), NaCl (up to 1 M). Interfering substances: >1 M urea, >1% SDS (precipitates), >10% glycerol, >5% ethanol/methanol, >1% formic acid, and basic solutions (will neutralize the acidic reagent). If sample contains high concentrations of interfering substances, use TCA precipitation or dialysis before assay. |
| Recommended Buffer System | Bradford Reagent: Coomassie G-250 (approximately 0.01% w/v) in 4.75% (w/v) phosphoric acid and 4.75% (v/v) methanol; BSA/BGG standard: 0.9% NaCl, 0.05% sodium azide. |
| Application Notes / Precautions | Pre-warm Bradford Reagent to RT before use; cold reagent may form dye aggregates causing high background. Mix reagent bottle well before pouring/pipetting — dye may settle during storage. Use BSA standard curve for most samples; use BGG for immunoglobulin-rich samples (serum, purified antibodies). Filter or centrifuge turbid samples (particulates scatter light and give falsely high readings). For detergent-containing samples, include a buffer-matched blank (lysis buffer without protein) to subtract background. The Bradford assay produces different color yields for different proteins (e.g., BSA vs lysozyme ratio ~2:1 at equal mass); for absolute quantification, use a standard protein similar to the target. For samples with >1% SDS, dilute to <0.1% SDS or use a detergent-compatible BCA assay instead. |
| Batch-to-Batch Consistency | BSA standard curve slope within +/-10% of reference lot; A595 of 1,000 ug/mL BSA within 0.4-0.6; blank A595 <0.3; detergent compatibility and inter-assay CV <5%. |
For research use only, not for clinical use.
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