Acetyl CoA Elisa Kits & Antibodies
ELISA (Enzyme-Linked Immunosorbent Assay) to measure Acetyl Coenzyme A (Acetyl CoA) levels:
Materials Needed:
- Coating antibody specific to Acetyl CoA
- Blocking buffer (e.g., BSA or milk)
- Acetyl CoA standards (prepared by serial dilution)
- Samples containing Acetyl CoA (e.g., cell lysates, tissue extracts, or biological fluids)
- Detection antibody specific to Acetyl CoA (conjugated to an enzyme)
- Substrate solution for the enzyme conjugate (e.g., TMB substrate)
- Stop solution (e.g., sulfuric acid or HCl)
- Wash buffer (e.g., PBS-Tween)
Protocol:
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Coating the Plate:
- Dilute the coating antibody specific to Acetyl CoA in coating buffer and add it to the wells of a microtiter plate. Incubate overnight at 4°C or for 2 hours at room temperature.
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Blocking:
- Wash the plate with wash buffer to remove unbound antibody.
- Block nonspecific binding sites on the plate by adding blocking buffer to each well. Incubate for 1 hour at room temperature or overnight at 4°C.
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Sample and Standard Addition:
- Prepare Acetyl CoA standards by serial dilution in sample diluent.
- Add standards and samples (diluted in sample diluent) to the plate wells. Include blank wells with sample diluent only.
- Incubate the plate for 2 hours at room temperature.
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Washing:
- Wash the plate 3-5 times with wash buffer to remove unbound analyte and antibodies.
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Detection:
- Add the detection antibody specific to Acetyl CoA (conjugated to an enzyme) to each well. Incubate for 1 hour at room temperature.
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Washing:
- Wash the plate again to remove unbound detection antibody.
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Signal Development:
- Add substrate solution to each well. Incubate for a specific time period (e.g., 15-30 minutes) until the color develops.
- Monitor color development and stop the reaction by adding stop solution when the desired intensity is reached.
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Measurement:
- Measure the absorbance of each well at the appropriate wavelength (typically 450 nm) using a microplate reader.
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Data Analysis:
- Generate a standard curve using the absorbance values of the standards to determine the concentration of Acetyl CoA in the samples.
- Calculate the concentration of Acetyl CoA in the samples based on the standard curve.
This protocol provides a basic guideline for performing an Acetyl CoA ELISA. Optimization of the assay conditions (e.g., antibody concentrations, incubation times)
