Glutamine synthetase (GS) is an enzyme that plays a crucial role in nitrogen metabolism by catalyzing the condensation of glutamate and ammonia to form glutamine. Glutamate + ATP + NH₃ → Glutamine + ADP + phosphate (Glutamine synthetase reaction). Glutamine synthetase utilizes ammonia produced by nitrate reduction, amino acid degradation, and photorespiration. The amide group of glutamate serves as a nitrogen source for metabolites in the glutamine synthesis pathway. Additional reactions may also occur via GS.
Experimental Principle
This kit employs a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). To microwells pre-coated with a mouse glutamine synthetase (GS) capture antibody, samples, standards, and biotin-labeled detection antibody are added sequentially, followed by HRP enzyme conjugate. After incubation and washing, the substrate TMB is used for color development. TMB is converted to blue by peroxidase (HRP) and to a final yellow under acidic conditions. The color intensity is positively correlated with the concentration of mouse glutamine synthetase (GS) in the sample. Absorbance (OD value) is measured at 450 nm using a microplate reader to calculate sample concentration.
Precautions
1. Strictly follow the specified time and temperature for incubation to ensure accurate results. All reagents must be brought to room temperature (20-25 °C) before use. Store reagents in the refrigerator immediately after use.
2. Improper washing may lead to inaccurate results. Ensure wells are thoroughly drained before adding substrate. Avoid allowing microwells to dry for extended periods during the procedure.
3. Clean residual liquid and fingerprints from the well bottoms, as these can affect OD values.
4. Substrate developing solution should be colorless; blue-tinted substrate must not be used.
5. Avoid cross-contamination between reagents and samples to prevent erroneous results.
6. Protect from direct strong light during storage and incubation.
7. Do not allow any reaction reagents to come into contact with bleaching agents or their vapors. Any bleaching compounds will inactivate the biological activity of the kit reagents.
8. Do not use expired products. Components from different catalog or batch numbers must not be mixed.
9. Recombinant proteins from sources other than this kit may not be recognized by the kit antibodies.
10. All samples should be handled with care and processed and tested in accordance with established protocols if there is a risk of disease transmission.
Sample Handling and Requirements
1. The kit’s detection range does not equate to the concentration range of the analyte in the sample. It is recommended to estimate the analyte concentration in the sample using relevant literature and confirm the actual concentration via a preliminary experiment. If the analyte concentration is too high or too low, dilute or concentrate the sample appropriately.
2. If the sample type is not listed in the manual, a preliminary experiment is recommended to verify detection validity.
3. Serum: Allow whole blood collected in serum separation tubes to stand at room temperature for 2 hours or overnight at 2-8 °C, then centrifuge at 1000 × g for 20 minutes and collect the supernatant. Alternatively, store the supernatant at −20 °C or −80 °C, avoiding repeated freeze-thaw cycles.
4. Plasma: Collect samples using EDTA or heparin as anticoagulant, centrifuge at 1000 × g for 15 minutes at 2-8 °C within 30 minutes of collection, and use the supernatant for testing. Alternatively, store the supernatant at −20 °C or −80 °C, avoiding repeated freeze-thaw cycles.
5. Tissue homogenate: Rinse tissue with pre-chilled PBS (0.01 M, pH=7.4) to remove residual blood (lysed red blood cells in homogenate will interfere with results). Weigh and mince the tissue. Place minced tissue and an appropriate volume of PBS (typically 1:9 w/v, e.g., 1 g tissue + 9 mL PBS; volume can be adjusted as needed and recorded) into a glass homogenizer, and grind on ice or using a homogenizer. For further cell lysis, sonicate or subject the homogenate to repeated freeze-thaw cycles. Centrifuge the homogenate at 5000 × g for 5-10 minutes and use the supernatant for testing.
6. Cell culture supernatant: Centrifuge at 1000 × g for 20 minutes and use the supernatant for testing. Alternatively, store the supernatant at −20 °C or −80 °C, avoiding repeated freeze-thaw cycles.
7. Cell lysate: Gently wash adherent cells with pre-chilled PBS, then digest with trypsin and collect cells by centrifugation at 1000 × g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with pre-chilled PBS, resuspend in 150-200 μL PBS per 1× 10⁶ cells (add protease inhibitors to PBS if recommended; reduce PBS volume if analyte is low), and lyse cells by repeated freeze-thaw or sonication. Centrifuge the extract at 1500 × g for 10 minutes at 2-8 °C and use the supernatant for testing.
8. Other biological samples: Centrifuge at 1000 × g for 20 minutes and use the supernatant for testing.
9. Sample appearance: Samples should be clear and transparent; remove any suspended matter by centrifugation.
10. Sample storage: If testing within 1 week, store samples at 4 °C. If testing is delayed, aliquot and store at −20 °C (for testing within 1 month) or −80 °C (for testing within 6 months), avoiding repeated freeze-thaw cycles. Hemolyzed samples should not be used as hemolysis interferes with results.
Sample Dilution Protocol
Estimate the sample concentration range in advance. If sample dilution is required, follow the protocol below:
1. 100-fold dilution: One-step dilution. Add 5 μL sample to 495 μL general diluent.
2. 1000-fold dilution: Two-step dilution. Add 5 μL sample to 95 μL general diluent (20-fold), then add 5 μL of the 20-fold dilution to 245 μL general diluent (50-fold); total dilution=1000-fold.
3. 100,000-fold dilution: Three-step dilution. Add 5 μL sample to 195 μL general diluent (40-fold), then add 5 μL of the 40-fold dilution to 245 μL general diluent (50-fold), then add 5 μL of the 2000-fold dilution to 245 μL general diluent (50-fold); total dilution = 100,000-fold.
In each step, use at least 3 μL for pipetting and do not exceed 100-fold dilution per step. Mix thoroughly after each dilution and avoid bubbles.
Required Self‑Provided Equipment
1. Microplate reader (450 nm)
2. High-precision pipettes and tips: 0.5-10 μL, 5-50 μL, 20-200 μL, 200-1000 μL
3. 37 °C incubator
4. Distilled or deionized water
Pre‑Test Preparation
1. Remove the kit from the refrigerator 10 minutes in advance and equilibrate to room temperature.
2. Preparation of standard gradient working solutions: Add 1 mL general diluent to the lyophilized standard, let stand for 15 minutes to fully dissolve, and mix gently (concentration = 20 ng/mL). Dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.31 ng/mL, 0 ng/mL.
Serial dilution method: Place 7 EP tubes, each containing 500 μL general diluent. Transfer 500 μL of the 20 ng/mL standard working solution to the first tube and mix to obtain 10 ng/mL. Repeat this step sequentially. The last tube serves as the blank and no liquid is transferred from the second-to-last tube.
3. Preparation of biotinylated detection antibody working solution: Centrifuge the 100× concentrated biotinylated detection antibody at 1000 × g for 1 minute 15 minutes before use. Dilute the 100× concentrate to 1× working concentration with general diluent (e.g., 10 μL concentrate + 990 μL diluent). Prepare fresh.
4. Preparation of enzyme conjugate working solution: Centrifuge the 100× concentrated enzyme conjugate at 1000 × g for 1 minute 15 minutes before use. Dilute the 100× concentrate to 1× working concentration with general diluent (e.g., 10 μL concentrate + 990 μL diluent). Prepare fresh.
5. Preparation of 1× washing solution: Add 10 mL of 20× washing solution to 190 mL distilled water (Crystals may form in the concentrated washing solution stored in the refrigerator; this is normal. Allow to reach room temperature and dissolve completely before preparation).
Procedure
1. Remove the required strips from the foil bag after equilibration to room temperature for 10 minutes. Seal the remaining strips in a resealable bag and return to 4 °C.
2. Sample addition: Add 100 μL of sample or standard of each concentration to the appropriate wells. Add 100 μL general diluent to the blank wells. Cover with a sealing film and incubate at 37 °C for 60 minutes. (Recommendation: Dilute test samples at least 1-fold with general diluent before adding to the plate to reduce matrix effects. Multiply the final calculated concentration by the dilution factor. Run all test samples and standards in duplicate.)
3. Add biotinylated detection antibody: Remove the plate, discard the liquid (no wash), and add 100 μL biotinylated detection antibody working solution to each well. Cover with a sealing film and incubate at 37 °C for 60 minutes.
4. Washing: Discard the liquid, add 300 μL 1× washing solution to each well, let stand for 1 minute, flick out the washing solution, and pat dry on absorbent paper. Repeat washing 3 times (or use a plate washer).
5. Add enzyme conjugate working solution: Add 100 μL enzyme conjugate working solution to each well. Cover with a sealing film and incubate at 37 °C for 30 minutes.
6. Washing: Discard the liquid and wash 5 times as described in step 4.
7. Add substrate: Add 90 μL TMB substrate to each well. Cover with a sealing film and incubate at 37 °C in the dark for 15 minutes.
8. Add stop solution: Remove the plate and add 50 μL stop solution to each well. Immediately measure the OD value at 450 nm.
Calculation of Experimental Results
Result Judgment:
1. Calculate the average OD value of duplicate standards and samples, then subtract the blank OD value to obtain the corrected OD value. Plot a standard curve using the four‑parameter logistic function on double‑logarithmic paper, with concentration on the x‑axis and OD value on the y‑axis.
2. If the sample OD value exceeds the upper limit of the standard curve, dilute the sample appropriately and re‑test; multiply the final concentration by the dilution factor.
Typical Data and Reference Curve
The following data and curve are for reference only. Users must establish their own standard curve based on experimental data.
| Concentration (ng/mL) | 20 | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.31 | 0 |
| OD value | 2.39 | 1.67 | 1.17 | 0.71 | 0.52 | 0.33 | 0.27 | 0.1 |
Corrected OD value
| 2.29 | 1.57 | 1.07 | 0.61 | 0.42 | 0.23 | 0.17 | - |
Note: This figure is for reference only. Calculate sample content based on the standard curve from each experiment.
Kit Performance
1. Repeatability: Intra‑assay CV< 10%; inter‑assay CV< 10%.
2. Recovery: Add three different concentrations of mouse GS to healthy mouse serum, plasma, and cell lysate, then calculate recovery.
Sample Type
| Range (%) | Average Recovery (%) |
| Serum (n=8) | 84-101 | 96 |
| Plasma (n=8) | 92-105 | 102 |
| Cell lysate (n=8) | 96-108 | 105 |
3. Linearity of dilution: Add high‑concentration mouse GS to four healthy mouse serum, plasma, and cell lysate samples, then dilute within the dynamic range of the standard curve to evaluate linearity.
Dilution Ratio
| Recovery (%) | Serum
| Plasma
| Cell lysate
|
| 1: 2 | Range
| 84-95 | 88-96 | 90-110 |
| 1: 2 | Average recovery
| 91 | 93 | 96 |
| 1: 4 | Range
| 89-103 | 87-108 | 105-115 |
| 1: 4 | Average recovery
| 94 | 98 | 109 |
Troubleshooting
If experimental results are unsatisfactory, photograph the color development, save experimental data, retain the used strips and unused reagents, and contact our technical support for assistance. You may also refer to the following:
| Problem Description | Possible Cause | Recommended Solution |
Poor standard curve linearity
| Incorrect standard dilution
| Ensure standards are dissolved and diluted as recommended
|
Poor standard curve linearity
| Inaccurate pipetting | Calibrate pipettes regularly and check tip sealing
|
Poor standard curve linearity
| Evaporation of reaction solution
| Seal the plate with a film
|
Poor standard curve linearity
| Inadequate washing
| Adequate washing times and sufficient washing solution volume
|
Poor standard curve linearity
| Foreign matter on well bottoms
| Clean well bottoms before reading
|
| Weak or no color development | Weak or no color development
| Insufficient incubation time Ensure full incubation time
|
| Weak or no color development | Incorrect incubation temperature
| Incubate at the recommended temperature
|
| Weak or no color development | Insufficient reagent volume
| Check pipettes and follow the protocol strictly
|
| Weak or no color development | Incorrect dilution
| Verify reagent dilution steps
|
| Weak or no color development | Inactivated enzyme conjugate
| Mix conjugate and substrate and check for color development
|
Low OD values
| Incorrect microplate reader settings | Check instrument wavelength |
Low OD values
| No stop solution added
| Add appropriate amount of stop solution
|
Low OD values
| Too long a wait before reading
| Read the plate promptly
|
Low OD values
| Sample concentration too high | Determine appropriate dilution via preliminary experiment
|
Low OD values
| Sample concentration too low | Determine appropriate dilution via preliminary experiment
|
| High background | Contaminated Substrate solution
| Replace Substrate solution
|
| High background | Excessive color development time
| Control color development time
|
| High background | Incorrect dilution of detection antibody or enzyme conjugate | Use recommended dilution method |
| High background | Inadequate washing
| Adequate washing times and sufficient washing solution volume |
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