1. Standard Curve Preparations: Colorimetric Assay: Dilute the Citrate Standard to 1 nM/µL by adding 10 µL of the Standard to 990 µL of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 µL into a series of standards wells on a 96 well plate. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nM/well of the Standard. Fluorometric Assay: Dilute the Citrate standard to 0.1 nM/µL by adding 10 µL of the standard to 990 µL of dH2O, mix well, then further dilute by adding 10 µL to 90 µL of dH2O. Add 0, 2, 4, 6, 8, 10 µL into a series of standards well on a 96-well plate. Adjust the volume to 50 µL/well to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nM/well.
2. Sample Preparation: Tissue (20 mg) or cells (2 x 10^6 ) should be rapidly homogenized with 100 µL of Citrate Assay Buffer. Centrifuge at 15,000 g for 10 minutes to remove cell debris. Enzymes in samples may interfere with the assay. We suggest deproteinizing samples using a perchloric acid/KOH protocol or 10 kd molecular weight cut off spin columns. Add 1-50 µL samples into duplicate wells of a 96-well plate and bring volume to 50 µL with Assay Buffer. We suggest testing several doses of your samples to ensure readings are within the standard curve range.
3. Develop: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µL Reaction Mix containing: Colorimetric Assay Fluorometric Assay Sample Bkgd Control* Sample Bkgd Control* Citrate Assay Buffer 44 µL 46 µL 44 µL 46 µL Citrate Enzyme Mix 2 µL ---- 2 µL ---- Developer 2 µL 2 µL 2 µL 2 µL Citrate Probe** 2 µL 2 µL 2 µL 2 µL *Samples may contain oxaloacetate or pyruvate which can generate a background and need to be subtracted from the sample background signal. **For the fluorometric assay, dilute 10X with DMSO to reduce fluorescent background.
4. Add 50 µL of the Reaction Mix to each well containing the Citrate Standard and test samples. Add 50 µL of the sample background control mix to background control wells.
5. Incubate for 30 minutes at room temperature, protect from light.
6. Measure OD at 570 nm or fluorescence at E x /E m 535/587nm.