Neurogranin ELISA Kit (NRGN) ELISA Kit
- Detection Method
- Method Type
- Competition ELISA
- Detection Range
- 39 pg/mL - 10000 pg/mL
- Minimum Detection Limit
- 39 pg/mL
- The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of human NRGN in Tissue Homogenate,Cell Lysate,Biological Fluids
- Sample Type
- Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
- Analytical Method
This assay has high sensitivity and excellent specificity for detection of Neurogranin (NRGN).
No significant cross-reactivity or interference between Neurogranin (NRGN) and analogues was observed.
- Cross-Reactivity (Details)
- No significant cross-reactivity or interference between Neurogranin (NRGN) and analogues was observed.
- 17 pg/mL
- Pre-coated, ready to use 96-well strip plate
- Plate sealer for 96 wells
- Standard Diluent
- Assay Diluent A
- Assay Diluent B
- Stop Solution
- Detection Reagent A
- Detection Reagent B
- TMB Substrate
- Wash Buffer (30 x concentrate)
- Instruction manual
- Material not included
- Microplate reader with 450 nm filter.
- Precision single or multi-channel pipettes and disposable tips.
- Eppendorf Tubes for diluting samples.
- Deionized or distilled water.
- Absorbent paper for blotting the microtiter plate.
- Container for Wash Solution
- Application Notes
- Limited by the current condition and scientific technology, we cannot completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
- The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
- Kits from different batches may be a little different in detection range, sensitivity and color developing time.
- Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
- Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
- There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
- Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
- Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
- Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
- Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.
Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.
Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.
Information on antibodies:
The provided antibodies and their host vary in different kits.
- Sample Volume
- 50 μL
- Assay Time
- 2 h
- This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to Neurogranin (NRGN) has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled Neurogranin (NRGN) and unlabeled Neurogranin (NRGN) (Standards or samples) with the pre-coated antibody specific to Neurogranin (NRGN). After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of Neurogranin (NRGN) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of Neurogranin (NRGN) in the sample.
- Reagent Preparation
- Bring all kit components and samples to room temperature (18-25°C) before use.
- Standard - Reconstitute the Standard with 0.5mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 50,000pg/mL. Please prepare 5 tubes containing 0.6mL Standard Diluent and produce a triple dilution series. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 50,000pg/mL, 16,666.7pg/mL, 5,555.6pg/mL, 1,851.9pg/mL, 617.3pg/mL, and the last EP tubes with Standard Diluent is the blank as 0pg/mL.
- Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate(2×) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. The prepared working dilution cannot be frozen.
- Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively (1:100).
- Wash Solution - Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1×).
- TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
- Making serial dilution in the wells directly is not permitted.
- Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37°C directly.
- Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10µL for once pipetting.
- The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
- If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until the crystals have completely dissolved.
- Distilled water is recommended to be used to make the dilution for reagents or samples. Contaminated water or container for reagent preparation will influence the detection result.
- Sample Collection
- Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, rinse tissues in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weigh before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work, too). Sonicate the resulting suspension with an ultrasonic cell disrupter or subject it to two freeze-thaw cycles to further break the cell membranes. Centrifugate the homogenates for 5 minutes at 5000 × g. Remove the supernate and assay immediately or aliquot and store at -20°C
Cell Lysate: Cells must be lysed before assaying according to the following directions. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). Wash cells three times in cold PBS. Resuspend cells in PBS (1×) and subject them to ultrasonication for 4 times (or Freeze cells at -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.) Centrifuge at 1500 × g for 10 minutes at 2 - 8°C to remove cellular debris.
Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
- Sample Preparation
- We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
- Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. Sample should be diluted by 0.01mol/L PBS(PH=7.0-7.2).
- If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
- Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
- Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g.antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
- Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
- Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.
- Assay Procedure
- Prepare all reagents, samples and standards,
- Add 50μL standard or sample to each well.
And then add 50μL prepared Detection Reagent A immediately.
Shake and mix. Incubate 1 hour at 37 °C,
- Aspirate and wash 3 times,
- Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
- Aspirate and wash 5 times,
- Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
- Add 50μL Stop Solution. Read at 450 nm immediately.
- Prepare all reagents, samples and standards,
- Calculation of Results
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between NRGN concentration in the sample and the assay signal intensity. Low levels of NRGN result in a high O.D value, while a high concentration of NRGN results in a low signal. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log graph paper, with NRGN concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, such as curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the log of concentration of the standard (Y-axis), although concentration is indeed the independent variable while O.D. value is the dependent variable. The O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects). This curve is provided for demonstration only. The customers should establish their own standard curve for each test conducted.
- Assay Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Neurogranin (NRGN) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Neurogranin (NRGN) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
- For Research Use only
- Precaution of Use
- The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
- Handling Advice
- The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.
- 4 °C
- Storage Comment
- For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C.
- For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
- For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.
- Expiry Date
- 6 months
Abner, Jicha, Shaw, Trojanowski, Goetzl: "Plasma neuronal exosomal levels of Alzheimer's disease biomarkers in normal aging." in: Annals of clinical and translational neurology, Vol. 3, Issue 5, pp. 399-403, 2016 (PubMed).
Goetzl, Kapogiannis, Schwartz, Lobach, Goetzl, Abner, Jicha, Karydas, Boxer, Miller: "Decreased synaptic proteins in neuronal exosomes of frontotemporal dementia and Alzheimer's disease." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 30, Issue 12, pp. 4141-4148, 2016 (PubMed).
- Abner, Jicha, Shaw, Trojanowski, Goetzl: "Plasma neuronal exosomal levels of Alzheimer's disease biomarkers in normal aging." in: Annals of clinical and translational neurology, Vol. 3, Issue 5, pp. 399-403, 2016 (PubMed).