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Analysis of Phosphorylated Compounds
Introduction:
The Fiske-Subbarow assay is an assay designed to measure levels of inorganic phosphate in biological samples. Fiske-Subbarow reducer reagent is used as the reducer component of the Fiske-Subbarow assay. Fiske-Subbarow reducer reagent is used as the reducer component of the Fiske-Subbarow assay.

Materials: * Water * KH2PO3 stock solution * Microplate * Micro pipette * Acidic Molybdate Reagent * Reducing agent * 6N HCL * Water bath * 3 Unknowns
Procedure:
* Prepare a set of phosphate dilutions from a 1mM KH2PO4 stock solution according to the following table: DILUTION | Volume (in uL) water | Volume (in uL) KH2PO3Stock solution | #1 | 300 | 0 | #2 | 250 | 50 | #3 | 200 | 100 | #4 | 100 | 200 | #5 | 0 | 300 |

* Prepare three columns of microwells * For each dilution prepared from the above table, transfer three times 75uL in separate wells of the microplate. * Obtain two unknowns (X1 and X2: 250uL of each) from your instructor or TA and transfer three times 75uL in separate wells of the microplate. * To all wells add:
25 uL acidic molybdate reagent
25 uL reducing agent
10 uL water * Keep at room temperature for 15 min. * Read the absorbance at about 600nm
Preparations for the stability evaluation of phosphorylates compounds * Obtain an unknown (X3; 1000 uL) containing unknown phosphorylated compounds from your instructor or TA. * From this sample, mix 500uL with either 100uL water or 100uL 6N HCL and cover. Store in your drawer till the next lab.

Acid stability test and phosphate sensitivity test of phosphorylated compounds * Obtain an unknown (X3; 1000 uL) containing unknown phosphorylated compounds from your instructor or TA * From this sample, mix 500uL with either 100uL water or 100uL phosphate solution and cover. Store in water bath at 37 degrees Celsius for 30 min. * Prepare a set of phosphate diltuions from a 1mM KH2PO4 stock solution as you did earlier in the experiment and collect the samples that were incubated with water or acid from earlier in the experiment. * Prepare three columns of microwells 1. For each dilution previously prepared 2. For each mixture prepared in the previous lab 3. For each mixture kept in the water bath

Transfer three times 75uL in separate wells of a microplate * To each well add:
25uL acidic molybdate reagent
25uL reducing agent
100uL water * Keep at room temperature for 15 min * Read the absorbance at about 600nm.

Calculations * For each standard dilution, calculate the concentration of phosphate present using the following equation:
[phosphate]= [Volume phosphate stock (in uL) * Concentration phosphate stock] / total volume (in uL) * For each standard solution, calculate the average absorbance and the standard deviation of the absorbance readings. * Contstruct a graph, phosphate concentration vs. average absorbance with Hi- Lo indicators of the standard deviation. * Perform a liner regression on the experimental data and show the regression equation on the graph * For the unknowns calculate the average absorbance and calculate the concentration of inorganic phosphate present using the linear regression equation obtained.

Results
Calibration and evaluation of the Fiske-Subbarow Assay

Acid Stability Test and Phosphate Sensitivity Test

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