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mAdiponectin DuoSet (1 KIT)
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View LargerMouse Adiponectin/Acrp30 DuoSet ELISA AdipoRon inhibits orthotopic pancreatic tumor growth and tumor proliferation in vivoMurine P-4313 and K-8484 cancer cells were orthotopically injected into syngeneic wildtype mice and allowed to grow for two weeks, after which mice were treated with vehicle or AdipoRon 5mg/kg/day for 2 weeks. (A) at endpoint, AdipoRon treated mice had a significantly reduced tumor size. (B) tumor proliferation measured by Ki67 positive tumor area per tumor section was significantly reduced after AdipoRon treatment. (C) no significant difference was detected in relative expression of AdipoR1 (white bars) and AdipoR2 (black bars) by quantitative real time PCR analysis between control and AdipoRon treated tumors. (D) ELISA analysis showed no significant difference in circulating serum adiponectin levels between the two groups of mice. (P-4313 filled symbols and K-8484 open symbols). Statistical analysis was performed using unpaired t-test (*P ≤ 0.05, **P ≤ 0.01). Image collected and cropped by CiteAb from the following open publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.19905), licensed under a CC-BY license. Not internally tested by R&D Systems.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) in PBS without carrier protein. Immediately coat a 96-well microplate with 100 µL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 µL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.
Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
PRECAUTION
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
Assay Procedure
- Add 100 µL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 µL of the Detection Antibody, diluted in Reagent Diluent (as recommended in the product datasheet), to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 µL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 µL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Mouse Adiponectin/Acrp30 DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Background: Adiponectin/Acrp30
Adiponectin (also known as Adipocyte complement-related protein of 30 kDa, Acrp30) is an adipocyte-derived protein with wide ranging paracrine and endocrine effects on metabolism and inflammation. It promotes adipocyte differentiation, fatty acid catabolism, and insulin sensitivity and is negatively correlated with obesity, type 2 diabetes, and atherogenesis. Adiponectin associates into 70 kDa noncovalently-linked trimers that are disulfide-linked into medium molecular weight hexamers and then into >300 kDa high molecular weight oligomers.