Enzyme Activity Assay Protocol
Generic protocol for Solarbio spectrophotometric enzyme activity assay kits. Optimized for microplate and cuvette-based measurements.
1. Equipment and Reagents Checklist
Required Equipment
| Equipment |
Specification |
Purpose |
| Spectrophotometer or microplate reader |
UV-capable (340 nm for NADH); visible range (405–660 nm) |
Absorbance measurement |
| Quartz cuvette (UV assays) |
1 cm path length, 100–500 μL capacity |
For 340 nm NAD(P)H measurements |
| Polystyrene cuvette (visible assays) |
1 cm path length |
For 400–660 nm chromogen assays |
| 96-well microplate |
Flat-bottom, clear |
High-throughput (200–300 μL well volume) |
| Precision micropipettes |
2–20 μL, 20–200 μL, 100–1000 μL |
Accurate reagent dispensing |
| Water bath or incubator |
37°C ± 0.5°C |
Temperature-controlled incubation |
| Microcentrifuge |
10,000–15,000×g |
Sample clarification |
| Homogenizer |
Glass Teflon homogenizer or bead mill |
Tissue/cell homogenization |
| Ice bucket |
— |
Keep samples and reagents cold |
| Timer |
±1 s accuracy |
Timing kinetic reactions |
| Vortex mixer |
— |
Mixing reaction components |
| Aluminum foil |
— |
Protect light-sensitive reagents (NADH, DCFH-DA) |
Consumables
| Item |
Recommended Specification |
| Microcentrifuge tubes |
0.5 mL, 1.5 mL, 2.0 mL (polypropylene) |
| Pipette tips |
Filter tips recommended for RNA/DNA work |
| Cuvettes |
Quartz (UV) or polystyrene (visible) |
| 96-well plates |
Clear flat-bottom, non-binding surface preferred |
2. Sample Preparation
Tissue Homogenization
| Step |
Action |
Time |
Notes |
| 1 |
Harvest and weigh tissue (0.05–0.1 g wet weight) |
2 min |
Blot dry on filter paper; use saline wash for blood-rich tissues |
| 2 |
Place in pre-cooled homogenization tube |
— |
Pre-chill tube on ice |
| 3 |
Add 10 volumes (w/v) of ice-cold kit extraction buffer |
1 min |
Volume = 1 mL per 0.1 g tissue |
| 4 |
Homogenize at 4°C (glass Teflon or bead mill) |
1–3 min |
Keep tube in ice water; avoid foaming |
| 5 |
Centrifuge 8,000–10,000×g at 4°C |
10 min |
Use refrigerated centrifuge |
| 6 |
Collect supernatant, transfer to fresh tube on ice |
1 min |
Avoid pellet; keep supernatant on ice |
| 7 |
Dilute if needed in kit-specific diluent |
— |
See individual kit instructions |
| 8 |
Proceed to assay |
— |
Complete within 2 h (or aliquot and freeze at -80°C) |
Cell Lysate Preparation
| Step |
Action |
Time |
Notes |
| 1 |
Harvest cells (1×10⁶ for most assays) |
5 min |
Trypsinize or scrape adherent cells |
| 2 |
Wash 2× with cold PBS (pH 7.4) |
5 min |
Centrifuge 300×g, 5 min, 4°C |
| 3 |
Resuspend pellet in 200–500 μL extraction buffer |
1 min |
Use kit-specific buffer |
| 4 |
Sonicate 3×10 s pulses on ice |
1 min |
Avoid foaming; 20% amplitude |
| 5 |
Centrifuge 10,000×g, 10 min, 4°C |
10 min |
Remove debris |
| 6 |
Collect supernatant |
1 min |
For cytosolic enzyme assays |
| 7 |
Assay same day or store at -80°C |
— |
Avoid repeated freeze-thaw |
Serum/Plasma
- Serum: Allow blood to clot 30 min at RT, centrifuge 1,000–2,000×g for 15 min at 4°C. Collect supernatant.
- Plasma: Use heparin or EDTA as anticoagulant. Centrifuge at 1,000–2,000×g for 10 min at 4°C.
- Dilution: Most serum assays require 1:2–1:10 dilution in PBS or kit diluent. For ALT/AST, use undiluted serum.
Quality Check: Hemolyzed samples yield invalid results for LDH, AST, and most RBC-rich enzyme assays. If hemolysis is visible (>0.1 g/L hemoglobin), reject the sample.
3. Reaction Setup
Generic Protocol for Kinetic (NADH-Coupled) Assays
| Step |
Component |
Volume (cuvette) |
Volume (96-well) |
| 1 |
Distilled water / buffer |
700 μL |
120 μL |
| 2 |
Substrate mix |
200 μL |
50 μL |
| 3 |
Coenzyme (NADH/NADPH) |
50 μL |
50 μL |
| 4 |
Sample |
50 μL |
30–50 μL |
| — |
Total |
1000 μL |
250–270 μL |
| 5 |
Mix gently by inversion or pipetting |
— |
| 6 |
Incubate at 37°C for 3 min (pre-read equilibration) |
— |
| 7 |
Read A₃₄₀ at t=0, then every 30 s for 3–5 min |
— |
| 8 |
Calculate ΔA/min |
— |
Generic Protocol for Endpoint (Chromogenic) Assays
| Step |
Component |
Volume (cuvette) |
Volume (96-well) |
| 1 |
Sample |
50–100 μL |
20–50 μL |
| 2 |
Substrate/buffer mix |
900 μL |
200 μL |
| 3 |
Mix, incubate at 37°C for specified time |
— |
|
| 4 |
Stop solution (if applicable) |
100 μL |
50 μL |
| 5 |
Read absorbance at kit-specific wavelength |
— |
|
Control Well Setup
| Well Type |
Contents |
Purpose |
| Blank |
Buffer + substrate (no sample) |
Zero instrument and measure background rate |
| Sample blank |
Sample + buffer (no substrate) |
Correct for endogenous absorbance |
| Positive control |
Known enzyme standard (e.g., 10 U/L) |
Validate kit performance |
| Sample |
Complete reaction with sample |
Measure enzyme activity |
| Inhibitor control |
Sample + substrate + inhibitor |
Confirm enzyme specificity |
4. Quality Checks During Protocol
Pre-Assay Quality Checks
| Check |
Acceptable Range |
Action if Out of Range |
| NAD(P)H initial A₃₄₀ |
0.8–1.5 AU |
If <0.8: add fresh NADH; if >1.5: dilute |
| Sample clarity |
Clear (no turbidity) |
Centrifuge at 12,000×g for 5 min |
| Blank rate (ΔA/min) |
<0.005/min |
Remake reagents if >0.005 |
| pH of reaction mix |
Within ±0.1 of kit specification |
Adjust with 1 M HCl or NaOH |
| Incubation temperature |
37°C ± 0.5°C |
Calibrate water bath |
During-Assay Checks
| Check |
Frequency |
Action |
| Linearity of ΔA/min |
Every 30 s for 5 min |
If R² < 0.98, reduce sample volume or concentration |
| ΔA/min value |
After first reading |
If >0.2/min: dilute sample 2×; if <0.005/min: increase sample 2× |
| Temperature uniformity |
At each read |
Maintain 37°C; use heated plate reader |
Post-Assay Checks
- Duplicate CV: If CV >10%, repeat the assay. Acceptable CV <5% for most kits.
- Standard curve R²: R² should be >0.99. Re-prepare standards if below.
- Positive control activity: Should be within 85–115% of expected value.
- Blank value: Should not differ from expected by >0.05 AU.
5. Calculation Methods
5.1 Kinetic Activity (NADH/NADPH) — Rate Measurement
Activity (U/L) = (ΔA/min) × V_total × 1000 / (ε × d × V_sample)
Where:
- ΔA/min = Average absorbance change per minute (linear portion)
- V_total = Total reaction volume (mL)
- ε = Molar extinction coefficient at 340 nm = 6.22 L·mmol⁻¹·cm⁻¹ for NADH
- d = Light path = 1 cm (cuvette) or 0.6 cm (200 μL/well in 96-well)
- V_sample = Sample volume (mL)
- 1000 = Conversion factor (mmol → μmol)
Example Calculation:
- ΔA/min = 0.042
- V_total = 1.0 mL (cuvette)
- ε = 6.22
- d = 1 cm
- V_sample = 0.05 mL
Activity = 0.042 × 1.0 × 1000 / (6.22 × 1 × 0.05) = 135 U/L
5.2 Endpoint Activity
Activity (U/L) = (A_sample − A_blank) × C_standard × Dilution / (A_standard − A_blank) × t
Where:
- A_sample = Sample absorbance
- A_blank = Blank absorbance
- C_standard = Standard concentration (from included calibrator)
- Dilution = Sample dilution factor
- t = Reaction time (min, for per-min units)
5.3 Specific Activity (per mg protein)
Specific activity (U/mg protein) = Activity (U/L) / Protein concentration (mg/mL)
See Protein Quantification Kits for protein quantification methods.
6. Troubleshooting Table
| Issue |
Cause |
Solution |
Prevention |
| No activity detected (ΔA ~ 0) |
Sample not added; enzyme inactive or denatured |
Check pipetting; use fresh sample; add protease inhibitors |
Keep samples on ice; add protease inhibitors to extraction buffer |
| Activity too high (ΔA/min > 0.2) |
Sample too concentrated |
Dilute sample 2–10 fold; reduce sample volume |
Pre-dilute based on expected activity |
| Activity decreases over time (curving) |
Substrate depleted; product inhibition; NADH consumed |
Reduce sample volume; shorten measurement time |
Verify linear phase; use 2× substrate concentration |
| Negative rate (absorbance increases) |
Wrong direction; interfering enzyme; NADPH production |
Confirm wavelength; check kit direction |
Read kit protocol carefully; include sample blank |
| Irregular reading (scatter, spikes) |
Bubbles in solution; precipitate formation |
Tap plate; centrifuge sample; filter reagent |
Mix gently; avoid SDS precipitation in Bradford |
| High blank rate (ΔA > 0.01/min) |
NADH auto-oxidation; contaminated reagents |
Prepare fresh NADH solution; use fresh buffer |
Prepare NADH immediately before use; protect from light |
| Equal test and control (no inhibition) |
Inhibitor inactive; compound inefficient |
Check inhibitor concentration; increase inhibitor |
Verify inhibitor stock; test dose-response |
| Ran out of NADPH before kinetic read |
Sample activity too high |
Dilute sample; use more NADPH (0.3 mM final) |
Check sample dilution in pilot experiment |
| Sample blank shows high A |
Hemoglobin, pigments, or turbidity |
Centrifuge at higher speed; dilute sample |
Use matched sample blank for each sample |
| Standard curve nonlinear |
Pipetting error; chromogen instability |
Repeat with fresh standards; use fresh chromogen |
Calibrate pipettes; prepare new standards each time |
7. Optimization Notes
For Different Sample Types
| Sample Type |
Modification |
Reason |
| Adipose tissue |
Use higher detergent (0.5% deoxycholate) |
High lipid content causes turbidity |
| Muscle tissue |
Dilute 1:5–1:20 (high enzyme content) |
High metabolic enzyme activity |
| Plant tissues |
Add 1–2% PVP (polyvinylpyrrolidone) |
Remove phenolic interference |
| Bacterial lysates |
Add lysozyme (1 mg/mL) + sonication |
Complete cell lysis |
| Culture supernatant |
Concentrate 10× (Amicon 10K MWCO) |
Low enzyme concentration |
| Hemolyzed serum |
Reject sample (use fresh collection) |
RBC enzymes inflate activity |
Scaling Instructions
| Format |
Cuvette (1 mL) |
96-well (250 μL) |
384-well (100 μL) |
| Sample volume |
50–200 μL |
10–50 μL |
5–20 μL |
| Total volume |
1000 μL |
250 μL |
100 μL |
| Path length |
1.0 cm |
~0.6 cm |
~0.4 cm |
| Sensitivity |
Reference |
~60% of cuvette |
~40% of cuvette |
For microplate assays, multiply calculated activity by factor (1.0 / actual path length in cm). Accurate path length depends on total volume; calibrate using water absorbance at 977 nm.
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