User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/11/13

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ADA Kinetic Assay Preparations

  • A weight of 0.6702 g of sodium phosphate dibasic was dissolved in 50 mL of water to obtain a molarity of 0.05 M. The pH of the solution was adjusted to pH 7.4.


.050 L of water × \frac{0.05 mol}{1L} = .0025 mol of Na2HPO4 × \frac{268.07 g}{1 mol} = 0.6702 g


  • The pH was was adjusted to 7.4 by the addition of 2 drops of 12 M HCl.
  • To obtain 1 mM inosine, 1.5 mg of the solid was dissolved in 1 mL buffer. This was further diluted by collecting 89.3 µL of the 1.5 mg/ml inosine into 5 mL of the sodium phosphate buffer.


.0015 g of inosine × \frac{1 mol}{268.2 g} = .000005596 mol ÷ .001 L = .005596 M = 5.596 mM


5.596 mM (V1)= 0.1 mM (5 mL)

V1 = 0.08934 mL = 89.3 μL in 5 mL of buffer

  • 3 mM adenosine was prepared by dissolving 0.0082 g of the solid into 10 mL sodium phosphate buffer. The stock concentration of adenosine was 3.07 mM.


.0082 g of adenosine × \frac{1  mol}{267.24  g} = 3.06840 × 10-5 ÷ .010 L = .00307 M = 3.07 mM

UV-visible scans of Reagents

  • Mody and Nagle condcuted the runs for the UV-visible scans of ADA, adenosine, and inosine to verify the absorbance peaks for each.
  • From the ADA Activity Assay protocol, it was specified to monitor the absorbance of each reagent at wavelengths 235 and 265.
  • 1 mL of inosine was transferred to a cuvette. The final concentration of inosine in the cuvette was 1 mM.
  • 10 μL of adenosine was diluted to 990 μL of the sodium phosphate buffer in a cuvette. The final concentration of adenosine was .0307 mM.

M1V1 = M2V2

(3 mM)(10 μL) = M2 (1000 μL)

M2 = .0307 mM

Image:SmallerAdenoino1113.png


  • ADA was diluted as follows:

3 μL of ADA × 65 μM = M2 (1000 μL)

M2 = .195 μM of ADA


500 μL × .195 μM = M2 (1000 μL)

M2 = .0975 μM = 97.5 nM

  • 2 additional 500 μL dilutions of 97.5 nM to 1000 μL were executed using the dilution equation.

Second dilution M = 48.8 nM

Final Concentration = 24.38 nM


Image:ADA.png


Beer's Law

  • Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine and inosine Their absorbance are listed on the table below.
  • By manipulating Beer's Law, the molar absorptivity was calculated from the absorbance and concentration of the substances.
\frac{\epsilon A}{bc}
Wavelength Adenosine Inosine
Absorbance at 235.57.14
Absorbance at 265.4.6


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