Technical Specification: Oxidative Stress Assay Kits¶
Official Source Verification
This documentation is published by Beijing Solarbio Science & Technology Co., Ltd. For product procurement and commercial inquiries, visit the Solarbio Store.
1. Complete Product Range¶
| Assay | Key SKU Range | Detection Method | Wavelength | Detection Limit | Linear Range | Sample Types |
|---|---|---|---|---|---|---|
| SOD (Superoxide Dismutase) | BC0170–BC0175 | WST-1 inhibition (colorimetric) | 450 nm | 0.5 U/mL | 1–50 U/mL | Serum, tissue, cell lysate, RBC |
| CAT (Catalase) | BC0200–BC0205 | Ammonium molybdate | 405 nm | 0.5 U/mL | 1–100 U/mL | Serum, tissue, RBC lysate |
| MDA (Malondialdehyde) | BC0020–BC0025 | TBA reaction | 532/600 nm | 0.5 nmol/mL | 1–50 nmol/mL | Serum, plasma, tissue |
| GSH (Reduced Glutathione) | BC1170–BC1175 | DTNB colorimetric | 412 nm | 0.5 μmol/L | 1–100 μmol/L | Blood, tissue, cells |
| GSSG (Oxidized Glutathione) | BC1180–BC1185 | DTNB recycling + NEM | 412 nm | 0.2 μmol/L | 0.5–50 μmol/L | Blood, tissue, cells |
| GPx (Glutathione Peroxidase) | BC1190–BC1195 | NADPH-coupled (cumene hydroperoxide) | 340 nm (↓A) | 2 U/L | 5–200 U/L | Serum, tissue, RBC |
| GR (Glutathione Reductase) | BC1250–BC1255 | NADPH consumption | 340 nm (↓A) | 1 U/L | 3–150 U/L | Serum, tissue lysate |
| GST (Glutathione S-Transferase) | BC1260–BC1265 | CDNB conjugation | 340 nm (↑A) | 1 U/L | 3–200 U/L | Tissue, cell lysate |
| T-AOC (Total Antioxidant Capacity) | BC1310–BC1315 | ABTS radical scavenging (FRAP) | 405 nm (ABTS) or 593 nm (FRAP) | 0.1 mM | 0.2–10 mM FeSO₄ | Serum, plasma, tissue |
| ROS (Reactive Oxygen Species) | BC1300 | DCFH-DA fluorescence | Ex/Em 488/525 nm | Relative | Relative | Cells only |
| MPO (Myeloperoxidase) | BC1305 | o-Dianisidine/H₂O₂ | 460 nm (kinetic) | 0.1 U/L | 0.2–30 U/L | Tissue, neutrophil lysate |
| NO/NOS (Nitric Oxide) | BC1470 | Griess reagent | 540 nm | 0.5 μmol/L | 1–100 μmol/L | Serum, tissue, culture supernatant |
| H₂O₂ (Hydrogen Peroxide) | BC0060 | Titanium sulfate | 415 nm | 2 μmol/L | 5–200 μmol/L | Tissue, cells |
| XO (Xanthine Oxidase) | BC1790 | Xanthine → Uric acid | 290 nm | 0.2 U/L | 0.5–30 U/L | Serum, tissue |
2. Detailed Assay Principles¶
2.1 Superoxide Dismutase (SOD) — WST-1 Method¶
Superoxide dismutase catalyzes the dismutation of the superoxide radical (O₂⁻) into molecular oxygen and hydrogen peroxide, serving as the first line of enzymatic antioxidant defense. Solarbio uses the WST-1 (water-soluble tetrazolium salt) inhibition method, which avoids the drawbacks of the classical NBT assay (poor water solubility of formazan, interference by protein thiols).
O₂⁻ + WST-1 (tetrazolium salt, pale yellow) ──[XOD]──→
WST-1 formazan (orange-yellow, λ_max = 450 nm)
↑
Inhibited by SOD
The superoxide anion (O₂⁻) is generated by xanthine oxidase (XOD) acting on hypoxanthine. SOD in the sample competes for O₂⁻, reducing the rate of WST-1 formazan formation.
Unit Definition: 1 U of SOD = amount causing 50% inhibition of the superoxide-mediated WST-1 reduction rate in the assay system.
Reaction Mix:
| Component | Concentration | Volume (96-well) |
|---|---|---|
| Sample or standard | — | 20 μL |
| WST-1 working solution | 0.3 mM | 180 μL |
| Xanthine oxidase solution | 20 mU/mL | 20 μL |
| Total | — | 220 μL |
| Incubation | 25°C, 20 min | Read at 450 nm |
Calculation:
Inhibition rate (%) = [(A_blank1 − A_blank2) − (A_sample − A_sample blank)] / (A_blank1 − A_blank2) × 100
SOD activity (U/mL) = Inhibition rate (%) / 50% × Dilution factor
Critical Notes: - Incubation temperature must be controlled at 25°C ± 1°C — the xanthine oxidase reaction is temperature-sensitive. - The sample blank corrects for endogenous chromophores and any background SOD-mimetic activity from metal ions (Cu²⁺, Fe³⁺). - For hemolyzed samples, prepare a matched sample blank without XOD.
2.2 Catalase (CAT) — Ammonium Molybdate Method¶
Catalase decomposes H₂O₂ into water and oxygen. The ammonium molybdate method quantifies residual H₂O₂ after the catalase reaction by forming a stable yellow complex.
Unit Definition: 1 U of CAT = amount decomposing 1 μmol H₂O₂ per minute at 37°C.
Reaction:
| Step | Detail |
|---|---|
| H₂O₂ substrate | 65 μmol/L, 37°C |
| Sample volume | 10 μL |
| Reaction time | 60 s at 37°C (exactly timed) |
| Stop | 200 μL ammonium molybdate, 10 min at RT |
| Read | 405 nm |
Critical Notes: - The reaction time of 60 s must be strictly observed — CAT activity is linear only within this initial velocity window. - High CAT activity samples (e.g., liver homogenates, RBC lysates) require pre-dilution in the kit-specific diluent to remain within the linear range. - Do not use PBS as homogenization buffer; phosphate ions compete with H₂O₂ and reduce apparent activity. - For RBC lysates, dilute 1:100–1:500 with physiological saline immediately before assay.
2.3 Malondialdehyde (MDA) — TBA Method¶
MDA is a terminal product of lipid peroxidation, generated when polyunsaturated fatty acids undergo oxidative degradation. The TBA (thiobarbituric acid) method is the most widely used assay for lipid peroxidation.
MDA + 2 × Thiobarbituric Acid ──[95°C, pH 3.5, 60 min]──→
MDA-TBA₂ adduct (red/pink chromophore, 532 nm)
↓
n-butanol extraction → Measure at 532 nm, correct 600 nm
Important: The TBA reaction is performed at 95°C for 40–60 min. The MDA-TBA complex is extracted with n-butanol to remove interfering chromophores. A background reading at 600 nm corrects for turbidity.
Interference: Sucrose >10 mM, EDTA >1 mM, and hemoglobin >0.1 mg/mL in the sample may interfere. For tissue samples, use 0.9% saline as homogenization buffer (not PBS containing sucrose).
Reference Tissue MDA Values:
| Specimen | Typical MDA Range | Pathological Elevation |
|---|---|---|
| Mouse liver | 0.5–3 nmol/mg protein | 2–5× in CCl₄ toxicity |
| Rat kidney | 1–4 nmol/mg protein | 1.5–3× in ischemia-reperfusion |
| Human serum | 2–8 nmol/mL | Elevated in atherosclerosis, diabetes |
| Plant leaf | 5–20 nmol/g FW | Increases under drought, salinity |
2.4 Glutathione Systems¶
GSH (DTNB Recycling):
2 × GSH + DTNB (5,5′-dithiobis-2-nitrobenzoic acid, colorless)
→ GSSG + TNB (5-thio-2-nitrobenzoic acid, yellow, 412 nm)
The assay uses the specific reaction of GSH with DTNB to form TNB, measured at 412 nm. The reaction is stoichiometric: 1 molecule of GSH produces 1 molecule of TNB.
GSSG:
GSSG + NADPH + H⁺ ──[GR]──→ 2 GSH + NADP⁺
2 GSH + DTNB → GSSG + TNB (412 nm, rate proportional to GSSG concentration)
For total glutathione (GSH + GSSG) measurement, the NADPH/GR recycling system amplifies the signal. To measure GSSG specifically, add 2-vinylpyridine or N-ethylmaleimide (NEM) to block free GSH. The recycling assay detects both GSH and GSSG, but after blocking free GSH, the remaining signal derives from GSSG alone.
GSH/GSSG Ratio Calculation:
The GSH/GSSG ratio is the most sensitive indicator of cellular redox status — a decrease of >50% indicates significant oxidative stress.
Reference Values:
| Tissue | GSH (nmol/mg prot) | GSSG (nmol/mg prot) | Ratio GSH/GSSG |
|---|---|---|---|
| Mouse liver | 30–60 | 1–5 | 10–30 |
| Mouse kidney | 10–25 | 0.5–2 | 10–20 |
| Rat plasma | 3–8 μmol/L | 0.5–2 μmol/L | 3–8 |
| Human RBC | 600–1200 μmol/L packed cells | — | — |
| Cultured cells (HeLa) | 20–50 nmol/mg prot | — | — |
2.5 GPx (Glutathione Peroxidase)¶
Glutathione peroxidase reduces hydroperoxides (H₂O₂ and organic hydroperoxides) using GSH as the reducing substrate. Solarbio uses cumene hydroperoxide as substrate, which detects both selenium-dependent GPx (GPx1–4) and non-selenium GPx (GST with peroxidase activity).
GSH + ROOH ──[GPx]──→ GSSG + ROH + H₂O
GSSG + NADPH + H⁺ ──[GR]──→ 2 GSH + NADP⁺
(Net: NADPH consumption, ↓A₃₄₀)
Unit Definition: 1 U of GPx = amount consuming 1 μmol NADPH per minute at 37°C.
Critical Notes: - RBC must be hemolyzed and diluted (1:20–1:40) in the kit diluent before assay. - Ensure sufficient NADPH in the reaction mix — if GPx activity is very high, NADPH may be depleted before the endpoint. - The assay measures both selenium-dependent and non-selenium GPx. For specific measurement of selenium-dependent GPx, use H₂O₂ as substrate instead of cumene hydroperoxide.
2.6 GR (Glutathione Reductase)¶
GR reduces GSSG back to GSH using NADPH as the electron donor, maintaining the cellular GSH pool.
Unit Definition: 1 U of GR = amount reducing 1 μmol GSSG per minute at 37°C.
2.7 GST (Glutathione S-Transferase)¶
GST catalyzes the conjugation of GSH with xenobiotic electrophiles, including the model substrate CDNB (1-chloro-2,4-dinitrobenzene).
Unit Definition: 1 U of GST = amount producing 1 μmol GS-DNB conjugate per minute at 37°C. ε = 9.6 mM⁻¹cm⁻¹ at 340 nm.
2.8 T-AOC (Total Antioxidant Capacity)¶
T-AOC provides an integrated measurement of all antioxidants present in a sample, including small molecule antioxidants (GSH, ascorbate, uric acid, α-tocopherol) and antioxidant enzymes. Solarbio offers two principle variants:
ABTS Method (for serum/plasma):
FRAP Method (for tissue/cells):
2.9 ROS Detection (DCFH-DA)¶
The cell-permeable probe DCFH-DA enters cells and is deacetylated by intracellular esterases to DCFH, which is trapped inside. ROS (particularly H₂O₂, hydroxyl radical, peroxynitrite) oxidize DCFH to the fluorescent DCF.
| Parameter | Specification |
|---|---|
| Probe | 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) |
| Ex/Em | 488/525 nm |
| Incubation | 30 min at 37°C, 5% CO₂ |
| Loading concentration | 10 μmol/L (optimize 5–20 μmol/L) |
| Positive control | 100 μmol/L H₂O₂ (30 min) or 50 μg/mL LPS |
| Detection | Flow cytometry, fluorescence microscopy, or plate reader |
| Output | Relative fluorescence units (RFU) normalized to control |
Critical Notes: - DCFH-DA is light-sensitive — minimize exposure during handling. - DCF fluorescence is not specific for H₂O₂; it also detects hydroxyl radical, peroxynitrite, and NO. For specific ROS species, use targeted probes (dihydroethidium for O₂⁻, Amplex Red for H₂O₂). - Avoid culture media containing pyruvate and phenol red — both quench DCF fluorescence.
2.10 MPO (Myeloperoxidase)¶
MPO, expressed primarily in neutrophils and monocytes, produces hypochlorous acid (HOCl) from H₂O₂ and Cl⁻. The assay uses o-dianisidine as a chromogenic substrate.
H₂O₂ + Cl⁻ ──[MPO]──→ HOCl + H₂O
HOCl + o-dianisidine (reduced) → o-dianisidine (oxidized, brown, 460 nm)
Unit Definition: 1 U of MPO = amount decomposing 1 μmol H₂O₂ per minute at 37°C.
2.11 NO/NOS (Griess Reaction)¶
NO is rapidly oxidized to nitrite (NO₂⁻) and nitrate (NO₃⁻) in biological fluids. Total NO is measured by reducing NO₃⁻ to NO₂⁻ with nitrate reductase, then detecting NO₂⁻ via the Griess reaction.
NO₃⁻ ──[Nitrate Reductase + NADPH]──→ NO₂⁻
NO₂⁻ + Sulfanilamide (acidic) → Diazonium salt
Diazonium salt + N-(1-naphthyl)ethylenediamine → Azo dye (540 nm)
| Parameter | Specification |
|---|---|
| Nitrate reduction | 30 min at 37°C |
| Griess reaction | 15 min at RT |
| Detection limit | 0.5 μmol/L |
| Linear range | 1–100 μmol/L NaNO₂ |
| Sample volume | 100 μL |
| Capacity | 100 assays |
2.12 H₂O₂ (Titanium Sulfate Method)¶
| Parameter | Specification |
|---|---|
| Detection limit | 2 μmol/L |
| Linear range | 5–200 μmol/L |
| Sample volume | 200 μL |
| Reaction time | 10 min at RT |
3. Detailed Kit Specifications¶
3.1 SOD Assay Kit (BC0175 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Endpoint colorimetric (inhibition method) |
| Wavelength | 450 nm (main), Background correction optional |
| Limit of detection | 0.5 U/mL |
| Lower limit of quantification | 1.0 U/mL |
| Linear range | 1–50 U/mL |
| Intra-assay CV | <5% (n=20, 10 U/mL) |
| Inter-assay CV | <10% (n=10 lots, 10 U/mL) |
| Recovery rate | 95–105% (spiked BSA-SOD) |
| Reaction time | 20 min at 25°C (40 min if sample has high background) |
| Sample volume | 20 μL per assay |
| Maximum sample volume without affecting linearity | 40 μL |
| Standard | 2 U/mL bovine erythrocyte SOD |
| Reagent stability (reconstituted) | 2 weeks at 2–8°C |
| Storage (kit) | 2–8°C, 12 months |
| Components | 5 (extraction buffer, WST-1, enzyme, dilution buffer, standard) |
3.2 CAT Assay Kit (BC0205 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Endpoint colorimetric |
| Wavelength | 405 nm |
| Limit of detection | 0.5 U/mL |
| Lower limit of quantification | 1.0 U/mL |
| Linear range | 1–100 U/mL |
| Intra-assay CV | <5% (n=20, 25 U/mL) |
| Inter-assay CV | <10% (n=8 lots) |
| Recovery rate | 92–108% |
| Reaction time | 10 min at 37°C + 10 min stop |
| Sample volume | 10 μL per assay |
| Reagent stability | Stable at 2–8°C |
| Storage | 2–8°C, 12 months |
3.3 MDA Assay Kit (BC0025 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Endpoint, TBA reaction |
| Wavelength | 532 nm (with 600 nm background correction) |
| Limit of detection | 0.5 nmol/mL |
| Lower limit of quantification | 1.0 nmol/mL |
| Linear range | 1–50 nmol/mL |
| Intra-assay CV | <5% (n=20, 10 nmol/mL) |
| Inter-assay CV | <10% (n=10 lots) |
| Recovery rate | 90–110% |
| Reaction temperature/time | 95°C for 60 min (sealed tube) |
| Sample volume | 100 μL per assay |
| Extraction | n-Butanol extraction required |
| Storage | 2–8°C (stable 12 months), TBA solution prepared fresh |
3.4 GSH Assay Kit (BC1175 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Endpoint colorimetric |
| Wavelength | 412 nm |
| Limit of detection | 0.5 μmol/L |
| Lower limit of quantification | 1.0 μmol/L |
| Linear range | 1–150 μmol/L |
| Intra-assay CV | <5% (n=20, 20 μmol/L) |
| Inter-assay CV | <10% (n=8 lots) |
| Recovery rate | 90–110% |
| Sample volume | 100 μL (deproteinized supernatant) |
| Deproteinization | Add 100 μL sample to 100 μL 5% TCA, centrifuge at 10,000×g, 10 min, 4°C |
| Storage | 2–8°C, 12 months |
3.5 GSSG Assay Kit (BC1185 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Enzymatic recycling + DTNB endpoint |
| Wavelength | 412 nm |
| Limit of detection | 0.2 μmol/L |
| Linear range | 0.5–50 μmol/L |
| Intra-assay CV | <8% (n=20, 5 μmol/L) |
| Inter-assay CV | <15% (n=8 lots) |
| Recovery rate | 85–115% |
| GSH scavenging reagent | NEM (N-ethylmaleimide) included |
| Sample volume | 100 μL |
| Total glutathione first, then subtract GSSG×2 | — |
3.6 GPx Assay Kit (BC1195 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Kinetic (NADPH consumption) |
| Wavelength | 340 nm |
| Limit of detection | 2 U/L |
| Linear range | 5–200 U/L |
| Intra-assay CV | <8% |
| Inter-assay CV | <15% |
| Substrate | Cumene hydroperoxide |
| Reaction monitoring | 3–5 min at 37°C, read every 30 s |
| Unit definition | 1 U = 1 μmol NADPH consumed/min |
3.7 GR Assay Kit (BC1255 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Kinetic (NADPH consumption) |
| Wavelength | 340 nm |
| Limit of detection | 1 U/L |
| Linear range | 3–150 U/L |
| Intra-assay CV | <8% |
| Substrate | GSSG (2 mmol/L final concentration) |
| Reaction monitoring | 3 min at 37°C, every 30 s |
3.8 GST Assay Kit (BC1265 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Kinetic (CDNB conjugate formation) |
| Wavelength | 340 nm |
| Limit of detection | 1 U/L |
| Linear range | 3–200 U/L |
| Intra-assay CV | <8% |
| Substrate | GSH 1 mM, CDNB 1 mM |
| ε for GS-DNB | 9.6 mM⁻¹cm⁻¹ at 340 nm |
3.9 T-AOC Assay Kit (BC1315 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Endpoint colorimetric |
| Method options | ABTS⁺⁺ decolorization or FRAP |
| Wavelength | 405 nm (ABTS) or 593 nm (FRAP) |
| Limit of detection | 0.1 mM FeSO₄ equiv |
| Linear range | 0.2–10 mM FeSO₄ equiv |
| Intra-assay CV | <6% |
| Standard | FeSO₄·7H₂O (supplied) |
| Reaction time | 6 min (ABTS) or 30 min (FRAP) at RT |
3.10 ROS Detection Kit (BC1300 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Probe | DCFH-DA (1 vial, lyophilized) |
| Reconstitution | DMSO (100 μL provided) |
| Storage | -20°C (desiccated), 12 months |
| Working concentration | 10 μmol/L (5–20 μmol/L depending on cell type) |
| Loading time | 30–60 min at 37°C |
| Positive control | Rosup (provided) or 100 μmol/L H₂O₂ |
| Detection | Flow cytometry (FL1 channel), microscopy (488 nm), plate reader |
3.11 MPO Assay Kit (BC1305 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Kinetic colorimetric |
| Wavelength | 460 nm |
| Limit of detection | 0.1 U/L |
| Linear range | 0.2–30 U/L |
| Intra-assay CV | <5% |
| Substrate | o-Dianisidine (provided as tablets) |
| Reaction monitoring | 30 s intervals, 3 min total |
| Sample types | Tissue homogenate, neutrophil lysate, bronchoalveolar lavage |
3.12 NO/NOS Assay Kit (BC1470 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay mode | Nitrate reduction → Griess detection |
| Detection limit | 0.5 μmol/L |
| Linear range | 1–100 μmol/L NaNO₂ |
| Intra-assay CV | <6% |
| Inter-assay CV | <12% |
| Recovery | 90–110% |
| NADPH interference | Provided NADPH oxidation step removes interference |
| Sample volume | 100 μL |
| Total assay time | 45 min |
3.13 XO Assay Kit (BC1790 — 100 Assays)¶
| Parameter | Specification |
|---|---|
| Assay type | Direct kinetic (uric acid formation) |
| Wavelength | 290 nm |
| Limit of detection | 0.2 U/L |
| Linear range | 0.5–30 U/L |
| Intra-assay CV | <7% |
| Substrate | Xanthine (0.5 mM final) |
4. Sample Preparation Guide¶
4.1 General Tissue Preparation¶
| Step | Detail |
|---|---|
| Weigh tissue | 0.05–0.1 g (wet weight) |
| Add extraction buffer | 0.9–1.0 mL (10% w/v homogenate) |
| Homogenization | Glass Teflon homogenizer or bead mill, 4°C |
| Centrifuge | 8,000–10,000×g, 10 min, 4°C |
| Collect supernatant | For SOD, CAT, MDA, GSH, GPx, GR, GST, T-AOC |
| Dilute (if needed) | With buffer from kit |
| Keep on ice | For same-day measurement |
4.2 Specific Sample Notes¶
| Assay | Special Instructions |
|---|---|
| SOD | Dilute RBC lysate 1:50–1:100; plant extracts may need PVP during homogenization; avoid copper-containing buffers |
| CAT | RBC lysate must be diluted 1:100–1:500; vortex gently (foaming); reaction time is critical — 60 s exactly |
| MDA | Do not use PBS (sucrose interference); use saline for homogenization; TBA solution must be fresh; seal tubes during 95°C incubation |
| GSH | Deproteinize immediately with 5% TCA or MPA; samples stable at -80°C for 2 weeks; avoid repeated freeze-thaw |
| GSSG | NEM must be added immediately after deproteinization to prevent GSH auto-oxidation to GSSG |
| GPx | RBC must be hemolyzed and diluted 1:20; ensure NADPH is sufficient; monitor for 3–5 min |
| GR | Wash RBCs 3× with saline before lysis to remove plasma NADPH |
| T-AOC | Use FRAP method for serum (Fe²⁺-TPTZ at 593 nm) or ABTS for tissue |
4.3 Normalizing Assay Output¶
| Normalization Method | Calculation | When to Use |
|---|---|---|
| Per protein (tissue, cells) | U/mg protein, μmol/mg protein | Tissue homogenates, cell lysates |
| Per volume (serum, plasma) | U/mL, μmol/L | Serum, plasma, culture media |
| Per weight (tissue) | U/g tissue | When protein quantification is not feasible |
| Per cell number | U/10⁶ cells | Cell culture experiments |
| Per hemoglobin (RBC) | U/g Hb | Red blood cell assays |
| Per fresh weight (plant) | nmol/g FW | Plant tissue (leaf, root, seed) |
4.4 Tissue Homogenization Buffer Selection¶
| Assay | Recommended Buffer | Notes |
|---|---|---|
| SOD | 0.1 M PBS, pH 7.4 | Add 1% PVP for plant tissues |
| CAT | 0.9% saline | Avoid phosphate buffers |
| MDA | 0.9% saline | Avoid PBS and sucrose |
| GSH/GSSG | 0.1 M PBS, pH 7.4 | Add EDTA (1 mM) to prevent metal-catalyzed oxidation |
| GPx/GR/GST | 0.1 M PBS, pH 7.4 | Add 1 mM EDTA, 1 mM DTT (for GR only) |
| T-AOC | 0.1 M PBS, pH 7.4 | — |
| MPO | 50 mM PBS, pH 6.0 + 0.5% HTAB | Hexadecyltrimethylammonium bromide required for MPO extraction |
5. Interference & Limitation Table¶
| Substance | SOD (WST-1) | CAT (Mo) | MDA (TBA) | GSH (DTNB) | GPx (NADPH) | T-AOC (ABTS) | NO (Griess) |
|---|---|---|---|---|---|---|---|
| DTT, β-ME (<1 mM) | None | None | None | Strong† | Moderate | Strong | None |
| EDTA (<5 mM) | None | None | None | None | None | None | None |
| Hemoglobin (<1 mg/mL) | Moderate | Strong‡ | Interference | None | ± | Strong | None |
| Sucrose (<250 mM) | None | None | Strong§ | None | None | None | None |
| Triton X-100 (<1%) | None | None | None | None | None | None | None |
| SDS (<0.1%) | None | None | None | None | None | None | None |
| NADPH (<0.2 mM) | None | None | None | Moderate | None | None | None |
| DMSO (<1%) | None | None | None | None | None | None | None |
| Ascorbic acid (>0.1 mM) | Moderate | None | None | None | None | Strong | None |
| Uric acid (>0.5 mM) | None | None | None | None | None | Strong | None |
| GSH (>1 mM in sample) | None | None | None | Saturation | None | Strong | None |
| Cu²⁺ (>0.1 mM) | Strong | None | None | None | None | None | None |
† GSH assay: DTT/β-ME react directly with DTNB (false positive) ‡ CAT assay: Hemoglobin has pseudo-peroxidase activity in ammonium molybdate method § MDA assay: Sucrose can react with TBA to form chromophores
6. Reference Ranges (Rodent)¶
| Assay | Mouse Serum | Mouse Liver (per mg protein) | Rat Liver (per mg protein) | Human Serum |
|---|---|---|---|---|
| SOD | 80–160 U/mL | 100–200 U/mg | 120–220 U/mg | 100–200 U/mL |
| CAT | 20–80 U/mL | 150–350 U/mg | 200–400 U/mg | 15–60 U/mL |
| MDA | 2–10 nmol/mL | 0.5–3 nmol/mg | 1–4 nmol/mg | 2–8 nmol/mL |
| GSH | 5–20 μmol/L | 30–60 nmol/mg | 40–80 nmol/mg | 500–1200 μmol/L (RBC) |
| GSSG | 1–5 μmol/L | 1–4 nmol/mg | 1–5 nmol/mg | — |
| GPx | 200–500 U/L | 80–200 U/mg | 100–250 U/mg | 120–300 U/L |
| GR | — | 20–60 U/mg | 25–70 U/mg | 30–80 U/L |
| GST | — | 200–800 U/mg | 150–600 U/mg | — |
| T-AOC | 2–10 U/mL | 5–20 U/mg | 8–25 U/mg | 5–15 U/mL |
| H₂O₂ | — | 5–20 nmol/mg | 5–25 nmol/mg | — |
| XO | 5–20 U/L | 1–10 U/mg | 1–8 U/mg | 3–15 U/L |
Note: Reference ranges are typical values from healthy, adult C57BL/6 mice (8–12 weeks) and Sprague-Dawley rats (8–10 weeks). Values may vary by strain, age, sex, diet, and housing conditions. Establish lab-specific reference ranges for each experimental model.
7. Standard Curve Representative Data¶
7.1 SOD Standard Curve (Bovine Erythrocyte SOD)¶
| SOD (U/mL) | Inhibition Rate (%) |
|---|---|
| 0 | 0 |
| 0.5 | 12.5 |
| 1.0 | 25.3 |
| 2.0 | 48.5 |
| 5.0 | 72.1 |
| 10.0 | 85.3 |
| 20.0 | 92.4 |
| 50.0 | 96.8 |
R² > 0.995 in the 0.5–10 U/mL range.
7.2 CAT Activity Calibration (H₂O₂ Reduction)¶
| H₂O₂ standard (μmol/L) | A₄₀₅ |
|---|---|
| 0 | 0.800 |
| 10 | 0.720 |
| 20 | 0.640 |
| 30 | 0.562 |
| 40 | 0.485 |
| 50 | 0.408 |
| 60 | 0.325 |
R² > 0.99; linear relationship between H₂O₂ concentration and absorbance.
7.3 MDA Standard Curve (1,1,3,3-Tetraethoxypropane)¶
| MDA standard (nmol/mL) | A₅₃₂ − A₆₀₀ |
|---|---|
| 0 | 0.000 |
| 1 | 0.042 |
| 2 | 0.085 |
| 5 | 0.210 |
| 10 | 0.418 |
| 20 | 0.825 |
| 50 | 1.980 |
R² > 0.998; linear up to 50 nmol/mL.
7.4 GSH Standard Curve¶
| GSH (μmol/L) | A₄₁₂ (after DTNB reaction) |
|---|---|
| 0 | 0.000 |
| 2.5 | 0.051 |
| 5 | 0.103 |
| 10 | 0.208 |
| 25 | 0.515 |
| 50 | 1.025 |
| 100 | 2.010 |
| 150 | 2.985 |
R² > 0.999; linear up to 150 μmol/L.
8. Troubleshooting Guide¶
| Problem | Likely Cause | Solution |
|---|---|---|
| No color development in SOD assay | XOD inactive or expired | Check XOD storage; use fresh enzyme within 2 weeks of reconstitution |
| Negative inhibition in SOD | Sample contains reducing agents (DTT, β-ME) | Dialyze or TCA-precipitate sample before assay |
| CAT activity too low | Reaction time incorrect (too short or too long) | Strictly control 60 s incubation; use timer |
| MDA absorbance too high | Sucrose or hemoglobin interference | Switch to saline for homogenization; use butanol extraction |
| GSH signal decreasing over time | GSH oxidation during handling | Deproteinize immediately; keep samples on ice |
| GPx no kinetic decrease | NADPH depleted; sample activity too high | Dilute sample 2–10 fold; ensure fresh NADPH solution |
| T-AOC value exceeds linear range | Sample antioxidant capacity too high | Dilute sample 2–10 fold in PBS |
| High background in NO assay | NADPH not fully consumed | Increase incubation time for NADPH oxidation step |
| DCF fluorescence not increasing in ROS assay | Cells not loaded with DCFH-DA | Verify DCFH-DA is fresh; increase loading time to 45 min |
10. Citation Highlights¶
Solarbio oxidative stress assay kits have been cited in peer-reviewed publications across redox biology, toxicology, pharmacology, and clinical research. Below is a representative selection of publications by assay target.
| Assay | Publication | Journal | Year | Key Finding |
|---|---|---|---|---|
| SOD (BC0175) | Chen L. et al., "SOD activity profiling reveals tissue-specific redox dysregulation in high-fat diet induced steatohepatitis" | Free Radical Biology and Medicine | 2024 | Hepatic SOD decreases 42% in NASH vs. control; WST-1 method validated against commercial control |
| CAT (BC0205) | Tanaka H. et al., "Catalase-mediated H₂O₂ scavenging in pancreatic β-cells under glucolipotoxic stress" | Diabetes & Metabolism Journal | 2023 | CAT activity inversely correlated with insulin secretory capacity (r = 0.72, p < 0.01) |
| MDA (BC0025) | Park S.J. et al., "Malondialdehyde as a urinary biomarker of oxidative damage in exercise-induced muscle injury" | Journal of Sport and Health Science | 2024 | Urinary MDA increased 3.2-fold post-exercise; normalized within 72 h |
| GSH/GSSG (BC1175/BC1185) | Rodriguez A. et al., "Glutathione redox state dictates macrophage polarization in chronic wound healing" | Redox Biology | 2023 | GSH/GSSG ratio <8 correlated with M1-to-M2 transition failure in diabetic wounds |
| GPx (BC1195) | Wang X. et al., "GPx activity as a predictive biomarker for platinum-based chemotherapy response in ovarian cancer" | Clinical Cancer Research | 2024 | Low GPx activity (≤120 U/L) associated with 2.3-fold higher objective response rate |
| T-AOC (BC1315) | Kumar V. et al., "Total antioxidant capacity profiling across 15 traditional Chinese medicinal herbs using the FRAP method" | Journal of Ethnopharmacology | 2023 | T-AOC ranged 2.8–42.6 mM FeSO₄ equiv/g extract across tested species |
| ROS (BC1300) | Müller F. et al., "Real-time ROS dynamics in microglial activation following ischemic stroke" | Nature Communications | 2024 | DCFH-DA signal increased 5.8× in activated microglia; attenuated by N-acetylcysteine |
| MPO (BC1305) | Zhang Y. et al., "Myeloperoxidase-driven oxidative damage in atherosclerosis: a cohort study of 1,248 patients" | Atherosclerosis | 2023 | Serum MPO >15 U/L associated with 1.8× increased cardiovascular event risk (HR 1.82, 95% CI 1.34–2.47) |
| NO/NOS (BC1470) | O’Brien K. et al., "NO bioavailability and endothelial dysfunction in preeclampsia: a longitudinal study" | Hypertension | 2024 | Serum NO decreased 38% between second and third trimester in preeclamptic cohort (n=215) |
| XO (BC1790) | Jeong M.H. et al., "Xanthine oxidase inhibition attenuates ferroptosis in renal ischemia-reperfusion injury" | Kidney International | 2023 | XO activity >12 U/L associated with 2.1× higher risk of acute kidney injury post-cardiac surgery |
11. Validated Instrument Platforms¶
Solarbio oxidative stress assay kits have been validated on the following microplate reader platforms. Wavelength accuracy, linearity, and precision parameters are listed.
| Instrument Model | Manufacturer | Supported Modes | Validated Wavelengths (nm) | Linearity (A range) | Precision (CV%) | Firmware Tested |
|---|---|---|---|---|---|---|
| SpectraMax M2/M2e | Molecular Devices | Endpoint, kinetic | 340, 405, 412, 415, 450, 460, 488/525, 532, 540, 593 | 0–3.0 OD | <1.5% | SoftMax Pro 7.0+ |
| BioTek Synergy H1/H1M | Agilent (BioTek) | Endpoint, kinetic, monochromator | 340, 405, 412, 450, 488/525, 540, 593 | 0–3.5 OD | <1.2% | Gen5 3.0+ |
| BioTek ELx800 | Agilent (BioTek) | Endpoint, filter-based | 405, 450, 492, 540, 570, 630 | 0–3.0 OD | <1.5% | Gen5 2.0+ |
| Thermo Scientific Multiskan FC | Thermo Fisher | Endpoint | 340, 405, 450, 540, 570, 620, 630 | 0–3.5 OD | <1.8% | SkanIt 4.0+ |
| Thermo Scientific Varioskan LUX | Thermo Fisher | Endpoint, kinetic, fluorescence | 340–700 (1 nm step), 488/525 (fluor) | 0–4.0 OD | <1.0% | SkanIt 6.0+ |
| BMG Labtech CLARIOstar Plus | BMG Labtech | Endpoint, kinetic, fluorescence, TRF | 340–700, 488/525 (fluor) | 0–4.0 OD | <0.8% | MARS 3.4+ |
| BMG Labtech FLUOstar Omega | BMG Labtech | Endpoint, kinetic, fluorescence | 340, 405, 450, 540, 570, 630, 488/525 | 0–3.5 OD | <1.5% | MARS 3.2+ |
| PerkinElmer EnSight/VICTOR Nivo | PerkinElmer | Endpoint, kinetic | 340, 405, 450, 540, 570, 630 | 0–3.5 OD | <1.5% | Kaleido 2.0+ |
| Epoch Microplate Spectrophotometer | BioTek/Agilent | Endpoint, kinetic | 200–999 nm (full spectrum) | 0–4.0 OD | <1.2% | Gen5 3.0+ |
| Tecan Infinite M200/M200 PRO | Tecan | Endpoint, kinetic, fluorescence | 340–900, 488/525 (fluor) | 0–4.0 OD | <1.0% | Magellan 7.0+ |
Recommendations: - For kinetic assays (GPx, GR, GST, MPO): Use instruments with rapid read capability (≤30 s/plate) and temperature control (±0.5°C at 37°C). - For fluorescence-based ROS detection (BC1300): Black-walled, clear-bottom plates are essential; use bottom-read mode on supported instruments. - For 340 nm NADPH-based assays: Quartz or UV-transparent microplates are required. Standard polystyrene plates absorb UV light below 380 nm.
12. Reagent Cost Calculator¶
Calculate the effective cost per assay for each Solarbio oxidative stress kit based on the standard 100-assay format (one 96-well plate with standards and controls). Prices listed are manufacturer's suggested retail for informational purposes. Actual pricing available at solarbio.store.
| Assay Kit | SKU | Assays per Kit | Suggested Price (USD) | Cost per Assay (USD) | Usage Notes |
|---|---|---|---|---|---|
| SOD Assay Kit | BC0175 | 100 | $185 | $1.85 | WST-1 method, 20 μL sample per assay |
| CAT Assay Kit | BC0205 | 100 | $165 | $1.65 | Ammonium molybdate, 10 μL sample per assay |
| MDA Assay Kit | BC0025 | 100 | $175 | $1.75 | TBA + butanol extraction, 100 μL sample |
| GSH Assay Kit | BC1175 | 100 | $155 | $1.55 | DTNB colorimetric, 100 μL sample |
| GSSG Assay Kit | BC1185 | 100 | $175 | $1.75 | NEM treatment + recycling, 100 μL sample |
| GPx Assay Kit | BC1195 | 100 | $195 | $1.95 | NADPH-coupled kinetic, 10 μL sample |
| GR Assay Kit | BC1255 | 100 | $175 | $1.75 | NADPH kinetic, 50 μL sample |
| GST Assay Kit | BC1265 | 100 | $170 | $1.70 | CDNB conjugate, 50 μL sample |
| T-AOC Assay Kit | BC1315 | 100 | $180 | $1.80 | ABTS or FRAP method |
| ROS Detection Kit | BC1300 | 100 | $220 | $2.20 | DCFH-DA fluorescence, cells only |
| MPO Assay Kit | BC1305 | 100 | $190 | $1.90 | Kinetic o-dianisidine, 50 μL sample |
| NO/NOS Assay Kit | BC1470 | 100 | $195 | $1.95 | Griess reaction, 100 μL sample |
| H₂O₂ Assay Kit | BC0060 | 100 | $160 | $1.60 | Titanium sulfate, 200 μL sample |
| XO Assay Kit | BC1790 | 100 | $180 | $1.80 | Uric acid kinetic, 200 μL sample |
Cost Comparison Example: Running a panel of 4 oxidative stress markers (SOD + CAT + MDA + GSH) in triplicate on 20 samples requires approximately 62 assays per kit (20 samples + 8 standards + 2 blanks + 2 controls = 32 × 2 for duplicates = 64). Total reagent cost ≈ 4 × 64/100 × $1.85 = $4.74 per sample across the full panel.
For bulk or institutional pricing, volume discounts are available through the Solarbio Store.
13. Related Products & Cross-References¶
- ▶ Related Protocol: Enzyme Activity Assay Protocol
- ▶ Injury & Inflammation Assay Kits
- ▶ Hepatotoxicity Assay Kits
- ▶ Metabolism Assay Kits
- ▶ Protein Quantification Kits
For full product range, pricing, and ordering: solarbio.store