Step 3 Prepare the samples as recommended in the package insert (e.g. diluting urine samples with water, or trichloroacetate precipitation of serum samples to remove protein). The diluted samples should contain 1 - 25 g Pi/mL. Pipet 2.5 mL of each into 12 x 75 mm glass tubes.Step 4 Add 0.5 mL acid molybdate solution and 0.125 mL Fiske & Subbarow solution to each sample and standard. Mix briefly with a vortex mixer. If using the microplate method, accurately transfer 250 L of each reaction mixture into the preassigned wells of the microplate.
Step 5 Incubate 10 minutes at room temperature. If using test tubes, read the absorbance of each tube in the cuvette port of the SPECTRAmax PLUS microplate spectrophotometer. For the reference reading, use the 0 standard. If desired, the samples can be transferred into cuvettes before reading. If using a microplate, place it in the drawer of the SPECTRA-max PLUS, then read the plate.
SIGMA KIT RESULTS
Figure 3 compares standard curves obtained using the same solutions, but read in 12 x 75 glass tubes and in a microplate. Both curves have excellent linearity from 1 mg/dL to 25 mg/dL based on the concentration of the original samples before dilution. (The actual concentration range of the diluted standards before addition of reagent is 125 g/mL.) The microplate standard curve is approximately 30% lower than the tube curve, reflecting the correspondingly shorter pathlength through the 250 L samples in the wells.
CHEN et al MATERIALS
1. SPECTRAmax PLUS microplate spectrophotometer system
2. Test tubes (12 x 75 mm)
3. Transparent microplates (e.g. Polystyrene) with lids
4. 1 cm cuvettes (if desired)
5. Samples (200-300 mL each, containing 0.1 to 5.0 g Pi/mL)
6. Pipettor and tips
7. 37 C in