Mouse Agouti Related Protein (AGRP) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDR-AGRP-Mu
The Mouse Agouti Related Protein (AGRP) ELISA Kit (RDR-AGRP-Mu) is an enzyme-linked immunosorbent assay for quantitative detection of AGRP in Mouse samples. This kit uses the Ready to Use ELISA format for colorimetric detection of natural AGRP in samples including serum, plasma, tissue homogenates, cell lysates and other biological fluids. The kit is easy to use and comes with a pre-coated microplate and all necessary reagents and buffers. The standard curve generated in the experiment is used to calculate the concentration of AGRP in samples. For research use only.
Product Specifications
Product Name
Mouse Agouti Related Protein (AGRP) ELISA Kit
Catalog Number
RDR-AGRP-Mu
Detection Range
78.1-5000pg/mL
Sensitivity
28pg/mL
Species Reactivity
Mouse
Species
Mus musculus Mouse
Assay Length
3 hours
Experimental Method
Sandwich
Detection Method
Colorimetric
Sample Type
serum, plasma, tissue homogenates, cell lysates and other biological fluids
Gene ID
11604
UniProtKB
P56473
Shelf Life
16 months
Synonyms
AGRTASIP2ARTAgouti-Related Peptide
Product Details
Intended Use
This kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of AGRP in mouse serum, plasma, tissue homogenates, cell lysates and other biological fluids.
Test Principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to AGRP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to AGRP. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain AGRP, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 ± 10 nm. The concentration of AGRP in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Application Data
Reagents and Materials Provided
| Reagents | Quantity | Reagents | Quantity |
|---|---|---|---|
| Pre-coated, ready to use 96-well strip plate | 1 | Plate sealer for 96 wells | 2 |
| Standard | 2 | Standard Diluent | 1 × 20 mL |
| Detection Solution A | 1 × 12 mL | TMB Substrate | 1 × 9 mL |
| Detection Solution B | 1 × 12 mL | Stop Solution | 1 × 6 mL |
| Wash Buffer (30× concentrate) | 1 × 20 mL | Instruction manual | 1 |
Materials Required but not Supplied
- Microplate reader with 450 ± 10 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.
Storage of the kits
- For unopened kits: All the reagents should be stored at -20°C upon receipt.
- For opened kits: Once the kit is opened, the remaining reagents still need to be stored according to the above storage conditions. In addition, return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
- For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this date.
- It is highly recommended to use the remaining reagents within 1 month of opening.
Sample Collection and Storage
- Serum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
- Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
- Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues should be rinsed in ice-cold PBS (0.01 mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed 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 also work). The resulting suspension should be sonicated with an ultrasonic cell disrupter or subjected to 2 freeze/thaw cycles to further break the cell membranes. Then, centrifuge the homogenates for 5 minutes at 5000 × g. Remove the supernatant and assay immediately or aliquot and store at ≤-20°C.
- Cell Lysates - 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 3 times in cold PBS.
- Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4 times (or Freeze cells at ≤-20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.).
- Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove cellular debris.
- Other biological fluids - Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
- Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and/or contamination.
- Noticeable hemolysis will affect antibody-antigen reactions. Samples with any sign of hemolysis are not acceptable for this assay.
- When performing the assay, bring samples to room temperature.
Reagent Preparation
- Bring all kit components and samples to room temperature (18-25°C) before use.
- Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 5000 pg/mL. Prepare 7 tubes containing 0.5 mL Standard Diluent and use the diluted standard to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Prepare a dilution series with 7 points; for example: 5000 pg/mL, 2500 pg/mL, 1250 pg/mL, 625 pg/mL, 312.5 pg/mL, 156.25 pg/mL, 78.125 pg/mL, and the last EP tube with Standard Diluent is the blank at 0 pg/mL.
- Detection Solution A and Detection Solution B - Detection Solutions A and B are already at the correct concentrations and do not need to be diluted further.
- Wash Solution - Dilute 20 mL of Wash Solution concentrate (30×) with 580 mL 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. Do not dump the residual solution back into the vial.
- Do not perform a serial dilution directly in the wells.
- Prepare standard within 15 minutes of performing the assay.
- Carefully reconstitute Standards according to the instruction, avoid foaming and mix gently until the crystals are completely dissolved.
- The reconstituted Standards 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 are completely dissolved.
- Any contaminated water or container used during reagent preparation will influence the detection result.
Assay Procedure Summary
- Prepare all reagents, samples and standards.
- Add 100 µL standard or sample to each well. Incubate 90 minutes at 37°C.
- Aspirate and add 100 µL Detection Solution A. Incubate 45 minutes at 37°C.
- Aspirate and wash 3 times.
- Add 100 µL Detection Solution B. Incubate 45 minutes at 37°C.
- Aspirate and wash 5 times.
- Add 90 µL Substrate Solution. Incubate 15-25 minutes at 37°C.
- Add 50 µL Stop Solution. Read at 450 nm immediately.
Calculation of Results
Average the duplicate readings for each standard, control and sample, then subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with AGRP concentration on the y-axis and absorbance on the x-axis. Using plotting software, (for instance, 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.
Product Data
Sensitivity
- The minimum detectable dose of AGRP is typically less than 28 pg/mL.
- The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Specificity
- This assay has high sensitivity and excellent specificity for detection of AGRP.
- No significant cross-reactivity or interference between AGRP and analogues was observed.
- Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between AGRP and all analogues, therefore, cross reactivity may still exist.
Precision
- Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level AGRP were tested 20 times on one plate, respectively.
- Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level AGRP were tested on 3 different plates, 8 replicates in each plate.
- CV (%) = SD/mean × 100
- Intra-Assay: CV<10%
- Inter-Assay: CV<12%
Stability
- The stability of ELISA 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 conditions.
- To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.
Research Areas