ELISA Rat - ANGPT2

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

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Found 3 matching solutions for this experiment

Upstream tips
-Store the unopened kit at 2-8 °C.
-Bring all reagents to room temperature before use.
-Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Protocol tips
-When mixing or reconstituting protein solutions, always avoid foaming.
-Substrate Solution should remain colorless until added to the plate.
-Keep Substrate solution protected from light.
-Stop Solution should be added to the plate in the same order as the Substrate Solution.
-Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with
the Substrate Solution.
Protocol tips
ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Samples in 96-well microplates were read by a Benchmark plus microplate spectrophotometer (Bio-Rad Laboratories, Inc.) within 20 min after the reaction was stopped. Test samples and standards were measured in duplicate. Microplate manager version 5.2.1 software (Bio-Rad Laboratories, Inc.), which was equipped with the spectrophotometer, automatically calculated sample concentrations according to corresponding standard curves with four-parameter logistics and log-logit function.
Upstream tips
-Store at 4°C for 6 months, at -20°C for 12 months.
-Avoid multiple freeze-thaw cycles.
-The standard solutions are best used within 2 hours.
Protocol tips
-The diluted Avidin-Biotin-Peroxidase Complex (ABC) solution should not be prepared more than 1 hour prior to the experiment.
-Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature.
-Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-labeled conjugate is used for detection.
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