Technical Specification: Plant Biology 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. Product Range¶
| Assay | SKU | Detection Method | Wavelength | Detection Limit | Linear Range | Sample Types |
|---|---|---|---|---|---|---|
| Chlorophyll Content Assay | BC0990 | Ethanol extraction + spectrophotometry | 665/649 nm | 0.5 mg/L | 1–50 mg/L chl a+b | Leaf, algae, chloroplast |
| Carotenoid Assay | BC0995 | Spectrophotometric (ethanol extract) | 470 nm | 0.2 mg/L | 0.5–20 mg/L | Leaf, fruit, flower |
| Proline Content Assay | BC0290 | Ninhydrin colorimetric | 520 nm | 1 μg/g FW | 2–50 μg/g FW | Leaf, root, seed |
| MDA (Plant) Assay Kit | BC0020-Plant | TBA reaction (plant-optimized) | 532/600 nm | 0.5 nmol/mL | 1–50 nmol/mL | Leaf, root, fruit |
| Nitrate Reductase Assay | BC0080 | Sulfanilamide-NED (nitrite detection) | 540 nm | 0.1 μmol NO₂⁻/h/g FW | 0.5–20 μmol NO₂⁻/h/g FW | Leaf, root |
| H₂O₂ Assay (Plant) | BC0060-Plant | Titanium sulfate | 415 nm | 1 μmol/g FW | 2–100 μmol/g FW | Leaf, root |
| Soluble Sugar Assay | BC5450 | Anthrone-sulfuric acid | 620 nm | 10 μg/g FW | 20–500 μg/g FW | Leaf, root, fruit |
| Starch Content Assay | BC0700 | Anthrone (amylase digestion) | 620 nm | 50 μg/g FW | 100–2000 μg/g FW | Leaf, seed, tuber |
| Total Phenolic Assay | BC1065 | Folin-Ciocalteu (GAE equiv) | 760 nm | 5 μg GAE/g | 10–200 μg GAE/g | Leaf, fruit, bark |
| Flavonoid Assay | BC1070 | AlCl₃ colorimetric (rutin equiv) | 510 nm | 10 μg RE/g | 20–500 μg RE/g | Leaf, flower, herb |
| Abscisic Acid (ABA) Assay | BC1188 | ELISA (competitive) | 450 nm | 1 ng/mL | 2–100 ng/mL | Leaf, xylem sap |
| Indole-3-Acetic Acid (IAA) Assay | BC1193 | ELISA (competitive) | 450 nm | 0.5 ng/mL | 1–50 ng/mL | Leaf, root, stem |
| Gibberellic Acid (GA₃) Assay | BC1197 | ELISA (competitive) | 450 nm | 1 ng/mL | 2–50 ng/mL | Leaf, seed |
2. Detailed Assay Principles¶
2.1 Chlorophyll — Spectrophotometric Method (BC0990)¶
Chlorophylls a and b are extracted in 95% ethanol and quantified spectrophotometrically using the specific absorption coefficients in ethanol.
Chl a (in 95% ethanol): λ_max = 665 nm (ε = 83.9 L·g⁻¹·cm⁻¹)
Chl b (in 95% ethanol): λ_max = 649 nm (ε = 48.5 L·g⁻¹·cm⁻¹)
Carotenoids: λ = 470 nm (combined signal)
Calculations (Lichtenthaler 1987, ethanol-based):
Chl a (mg/L) = 13.95 × A₆₆₅ − 6.88 × A₆₄₉
Chl b (mg/L) = 24.96 × A₆₄₉ − 7.32 × A₆₆₅
Chl a + b (mg/L) = 6.63 × A₆₆₅ + 18.08 × A₆₄₉
Carotenoids (mg/L) = (1000 × A₄₇₀ − 2.05 × Chl a − 114.8 × Chl b) / 245
| Parameter | Specification |
|---|---|
| Assay type | Endpoint spectrophotometric |
| Wavelengths | 665, 649, 470 nm |
| Limit of detection | 0.5 mg/L (each pigment) |
| Linear range | 1–50 mg/L |
| Intra-assay CV | <5% (n=20, 10 mg/L) |
| Inter-assay CV | <8% (n=6 lots) |
| Extraction solvent | 95% ethanol (v/v) |
| Extraction time | 24 h at 4°C (dark) |
| Minimum sample | 0.05 g fresh leaf |
| Solvent volume | 1 mL / 0.1 g tissue |
Protocol:
| Step | Detail |
|---|---|
| 1 | Weigh 0.1 g fresh leaf (avoid major veins) |
| 2 | Add 1 mL 95% ethanol, grind in mortar or bead mill |
| 3 | Transfer to tube, incubate in dark at 4°C for 24 h (or 60°C for 30 min for rapid extraction) |
| 4 | Centrifuge at 5000×g, 5 min |
| 5 | Read supernatant at 665, 649, 470 nm against ethanol blank |
| 6 | Calculate using equations above |
Reference Values:
| Plant Species | Chl a (mg/g FW) | Chl b (mg/g FW) | Chl a/b ratio |
|---|---|---|---|
| Arabidopsis (rosette) | 0.8–1.5 | 0.3–0.6 | 2.5–3.5 |
| Rice (flag leaf) | 2.0–4.0 | 0.6–1.2 | 3.0–3.5 |
| Maize (leaf) | 1.5–3.0 | 0.5–0.9 | 2.8–3.2 |
| Spinach (mature leaf) | 1.0–2.0 | 0.4–0.8 | 2.2–3.0 |
| Wheat (flag leaf) | 2.0–3.5 | 0.5–1.0 | 3.0–4.0 |
| Soybean (trifoliate) | 1.5–3.0 | 0.5–0.8 | 2.8–3.5 |
Stress-Induced Changes: - Drought: Chl ↓ 20–50%, Chl a/b ratio increases - Salt stress: Chl ↓ 30–60%, Chl a/b ratio may increase or decrease - Nitrogen deficiency: Chl ↓ primarily, Chl a/b ratio increases - Light stress: Chl a/b ratio decreases (increased antenna size)
Critical Note: Chlorophyll is photolabile. Perform all extraction steps in dim light or wrap tubes in aluminum foil. Do not use methanol or acetone without recalibrating equations (different absorption coefficients).
2.2 Carotenoid Assay (BC0995)¶
Carotenoids, including β-carotene and xanthophylls, are extracted together with chlorophyll and measured at 470 nm.
| Parameter | Specification |
|---|---|
| Wavelength | 470 nm |
| Limit of detection | 0.2 mg/L |
| Linear range | 0.5–20 mg/L |
| Reference range (leaf) | 0.2–0.8 mg/g FW |
Calculation (correcting for chlorophyll overlap at 470 nm):
2.3 Proline — Ninhydrin Colorimetric Method (BC0290)¶
Proline accumulates in plants under osmotic stress (drought, salinity, cold) as a compatible osmolyte. The assay uses the specific reaction of proline with ninhydrin in acidic conditions to form a red chromophore.
Proline + Ninhydrin ──[Glacial acetic acid, 100°C, 30 min]──→
Proline-ninhydrin complex (red, λ_max = 520 nm)
| Parameter | Specification |
|---|---|
| Assay type | Endpoint colorimetric |
| Wavelength | 520 nm |
| Limit of detection | 1 μg/g FW |
| Linear range | 2–50 μg/g FW |
| Intra-assay CV | <6% |
| Reaction temperature | 100°C (boiling water bath) |
| Reaction time | 30 min |
| Extraction | 3% sulfosalicylic acid (1 mL per 0.1 g tissue) |
| Solvent for extraction | Toluene (separates proline-ninhydrin complex) |
Protocol:
| Step | Detail |
|---|---|
| 1 | Homogenize 0.1 g tissue in 1 mL 3% sulfosalicylic acid |
| 2 | Centrifuge at 5000×g, 10 min |
| 3 | Collect 200 μL supernatant, add 200 μL ninhydrin reagent + 200 μL glacial acetic acid |
| 4 | Boil at 100°C for 30 min (sealed tube) |
| 5 | Cool on ice, add 1 mL toluene, vortex 15 s |
| 6 | Allow phase separation (15 min), read upper toluene phase at 520 nm |
Reference Values:
| Species | Control (μg/g FW) | Stress (μg/g FW) | Stress Condition |
|---|---|---|---|
| Arabidopsis | 5–15 | 30–80 | 200 mM NaCl, 48 h |
| Rice | 10–30 | 50–200 | Drought (15% PEG), 5 d |
| Wheat | 10–25 | 40–150 | 150 mM NaCl, 24 h |
| Maize | 5–20 | 30–120 | Drought, 7 d |
2.4 MDA (Plant) — TBA Method (BC0020-Plant)¶
The plant-optimized MDA kit accounts for plant-specific interferences (phenolics, sugars) that are less abundant in animal tissues.
MDA + 2 × Thiobarbituric Acid ──[95°C, pH 3.5, 40 min]──→
MDA-TBA₂ adduct (red, 532 nm; 600 nm correction)
| Parameter | Specification |
|---|---|
| Wavelength | 532 nm (600 nm correction) |
| Detection limit | 0.5 nmol/mL |
| Linear range | 1–50 nmol/mL |
| Intra-assay CV | <7% |
| Extraction buffer | 10% TCA (trichloroacetic acid) |
| Sample amount | 0.1 g tissue in 1 mL TCA |
Reference Values:
| Plant | Control (nmol/g FW) | Stress (nmol/g FW) |
|---|---|---|
| Rice leaf | 5–15 | 15–50 (salt stress) |
| Arabidopsis | 3–10 | 10–30 (drought) |
| Maize leaf | 5–20 | 20–60 (chilling) |
2.5 Nitrate Reductase (NR) Assay (BC0080)¶
Nitrate reductase catalyzes the first and rate-limiting step of nitrate assimilation, reducing NO₃⁻ to NO₂⁻.
NO₃⁻ + NADH ──[NR, 30°C, dark, 30 min]──→ NO₂⁻ + NAD⁺ + H₂O
NO₂⁻ + Sulfanilamide + NED ──[Acidic]──→ Azo dye (pink, 540 nm)
| Parameter | Specification |
|---|---|
| Assay type | In vitro activity |
| Wavelength | 540 nm |
| Detection limit | 0.1 μmol NO₂⁻/h/g FW |
| Linear range | 0.5–20 μmol NO₂⁻/h/g FW |
| Intra-assay CV | <8% |
| Incubation | 30°C, 30 min, dark |
| Substrates | KNO₃ (50 mM) + NADH (0.2 mM) |
Critical Note: NR is highly labile. Harvest tissue in mid-morning (light phase), process immediately at 4°C, and complete the assay within 2 h. The assay must be performed in the dark (light inactivates NR).
Reference Values:
| Species | NR Activity (μmol NO₂⁻/h/g FW) |
|---|---|
| Maize leaf (light) | 5–15 |
| Rice leaf | 3–10 |
| Arabidopsis | 2–8 |
| Spinach | 4–12 |
2.6 H₂O₂ Assay (BC0060-Plant)¶
| Parameter | Specification |
|---|---|
| Wavelength | 415 nm |
| Detection limit | 1 μmol/g FW |
| Linear range | 2–100 μmol/g FW |
| Extraction | Cold acetone or TCA |
| Reaction time | 10 min at RT |
Reference Values: Leaf H₂O₂ is typically 5–20 μmol/g FW under normal conditions and can increase to 30–100 μmol/g FW under stress.
2.7 Soluble Sugar — Anthrone Method (BC5450)¶
| Parameter | Specification |
|---|---|
| Wavelength | 620 nm |
| Detection limit | 10 μg/g FW |
| Linear range | 20–500 μg/g FW (glucose equivalents) |
| Extraction | 80% ethanol, 80°C, 30 min |
| Standard | Glucose (provided in kit) |
Reference Values:
| Species | Control (% DW) | Stress |
|---|---|---|
| Arabidopsis leaf | 1–3% | Up to 8% (drought) |
| Rice leaf | 3–8% | 5–15% (salt) |
| Maize leaf | 2–5% | 4–10% (cold) |
2.8 Starch Content (BC0700)¶
Starch is first digested to glucose by amylase/amyloglucosidase, then glucose is measured by the anthrone method.
Starch ──[Amylase, 60°C, 30 min]──→ Dextrins
Dextrins ──[Amyloglucosidase, 60°C, 30 min]──→ Glucose
Glucose ──[Anthrone/H₂SO₄]──→ Blue-green (620 nm)
| Parameter | Specification |
|---|---|
| Detection limit | 50 μg/g FW |
| Linear range | 100–2000 μg/g FW (glucose equiv) |
2.9 Total Phenolics — Folin-Ciocalteu (BC1065)¶
Phenolic OH + Folin-Ciocalteu reagent (Mo⁶⁺/W⁶⁺)
──[Na₂CO₃, alkalin]──→ Reduced Mo⁵⁺/W⁵⁺ (blue, 760 nm)
| Parameter | Specification |
|---|---|
| Wavelength | 760 nm |
| Detection limit | 5 μg GAE/g |
| Linear range | 10–200 μg GAE/g |
| Standard | Gallic acid (GAE) |
| Reaction time | 30 min at RT (dark) |
Reference Values:
| Plant Material | Total Phenolics (mg GAE/g DW) |
|---|---|
| Green tea leaves | 50–150 |
| Grape (skin) | 20–50 |
| Blueberry | 15–40 |
| Spinach leaf | 5–15 |
| Arabidopsis leaf | 3–10 |
2.10 Flavonoid Assay (BC1070)¶
| Parameter | Specification |
|---|---|
| Wavelength | 510 nm |
| Detection limit | 10 μg RE/g |
| Linear range | 20–500 μg RE/g |
| Standard | Rutin (RE) |
| Reaction time | 10 min at RT |
2.11 Plant Hormone ELISAs (ABA, IAA, GA₃)¶
These competitive ELISA kits use a HRP-conjugated hormone tracer and TMB substrate.
| Parameter | ABA (BC1188) | IAA (BC1193) | GA₃ (BC1197) |
|---|---|---|---|
| Assay type | Competitive ELISA | Competitive ELISA | Competitive ELISA |
| Detection limit | 1 ng/mL | 0.5 ng/mL | 1 ng/mL |
| Linear range | 2–100 ng/mL | 1–50 ng/mL | 2–50 ng/mL |
| Intra-assay CV | <8% | <9% | <9% |
| Inter-assay CV | <15% | <15% | <15% |
| Recovery (spiked tissue extract) | 85–115% | 80–120% | 85–115% |
| Cross-reactivity | ABA >95% | IAA >95% | GA₃ >90% |
| Sample type | Leaf (0.5 g), xylem sap (100 μL) | Leaf (0.5 g), root (0.5 g) | Leaf (0.3 g), seed (0.1 g) |
| Extraction | 80% MeOH + BHT | 80% MeOH | 80% MeOH |
| Incubation time | 2 h + 15 min TMB | 2 h + 15 min TMB | 2 h + 15 min TMB |
Sample Extraction for Hormones: Plant tissue (0.3–0.5 g) is extracted in 80% methanol containing 10 mg/L BHT (butylated hydroxytoluene) at 4°C for 12–16 h. The extract is centrifuged, dried under N₂, and reconstituted in assay buffer. Further purification by C18 SPE cartridge is recommended for tissues with high pigment or phenolic content.
3. Sample Preparation Guide¶
| Assay | Recommended Tissue Amount | Extraction Solvent | Special Notes |
|---|---|---|---|
| Chlorophyll | 0.1 g leaf | 95% ethanol, 24 h, 4°C, dark | Avoid major veins; process in dim light |
| Carotenoids | 0.1 g leaf | Same as chlorophyll (read at 470 nm) | Co-extracted with chlorophyll |
| Proline | 0.1 g leaf/root | 3% sulfosalicylic acid | Use fresh tissue; avoid drying |
| MDA | 0.1 g leaf | 10% TCA (plant-optimized) | Do not use PBS or sucrose-containing buffers |
| NR | 0.2 g leaf | In situ in assay buffer (no extraction) | Must perform in dark; process within 2 h |
| H₂O₂ | 0.1 g leaf | Cold acetone or 5% TCA | Extract quickly; H₂O₂ degrades rapidly |
| Soluble Sugar | 0.1 g leaf (DW) | 80% ethanol, 80°C | Use oven-dried (80°C, 48 h) or fresh tissue |
| Starch | Residue from sugar extraction | DMSO + heat | Use pellet from sugar extraction |
| Total Phenolics | 0.1 g DW | 70% methanol, sonicate | Protect from light; use dark glass vials |
| ABA | 0.5 g leaf | 80% MeOH, 4°C, 12 h | C18 SPE recommended for clean-up |
4. Troubleshooting Guide¶
| Problem | Likely Cause | Solution |
|---|---|---|
| Chlorophyll A₆₆₅ exceeds 2.0 | Tissue sample too large | Reduce sample to 0.05 g; dilute extract 1:2 |
| Proline blank is colored | Ninhydrin reagent degraded or overheated | Prepare fresh ninhydrin; reduce heating time |
| MDA absorbance decrease after extraction | TBA chromophore instability | Read within 30 min after extraction |
| NR activity zero | Tissue harvested in dark phase | Harvest 2–4 h into light cycle |
| H₂O₂ blank high | Titanium reagent hydrolyzed | Prepare fresh H₂SO₄/Ti solution weekly |
| Anthrone blank is green | Sulfuric acid concentration incorrect | Ensure H₂SO₄ is >95% and cooled before use |
| Hormone binding below B₀ | Extract contains organic solvents | Evaporate MeOH completely and reconstitute in buffer |
5. Stress Marker Panel Recommendations¶
| Stress Type | Primary Markers | Secondary Markers | Expected Change |
|---|---|---|---|
| Drought | Proline (⬆), MDA (⬆), Soluble Sugars (⬆) | Chlorophyll (⬇), NR (⬇), ABA (⬆) | Proline 3–10×; MDA 2–3×; Sugars 1.5–3× |
| Salinity | Proline (⬆), MDA (⬆), H₂O₂ (⬆) | Chlorophyll (⬇), Carotenoids (variable) | Proline 5–20×; H₂O₂ 2–5× |
| Cold | Proline (⬆), Soluble Sugars (⬆), MDA (⬆) | Chlorophyll (⬇), Phenolics (⬆) | Sugars 2–4×; Proline 2–5× |
| Heat | MDA (⬆), H₂O₂ (⬆), Proline (⬆) | Chlorophyll (⬇), NR (⬇) | MDA 2–4×; H₂O₂ 2–3× |
| Heavy metal (Cd) | MDA (⬆), H₂O₂ (⬆), GSH (⬆) | Chlorophyll (⬇), Proline (⬆) | MDA 2–5×; GSH 1.5–3× |
| UV-B | Phenolics (⬆), Flavonoids (⬆), MDA (⬆) | Chlorophyll (⬇), Carotenoids (⬆) | Phenolics 2–5×; UV-absorbing compounds |
| Pathogen defense | Phenolics (⬆), H₂O₂ (⬆), ABA (⬆) | Flavonoids (⬆), Salicylic acid | H₂O₂ burst 2–10×; phenolics 2–3× |
6. Related Products & Cross-References¶
- ▶ Oxidative Stress Assay Kits
- ▶ Metabolism Assay Kits
- ▶ Enzyme Activity Assay Kits
- ▶ Related Protocol: Enzyme Activity Assay Protocol
For full product range, pricing, and ordering: solarbio.store