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

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(UV-visible scans of Reagents)
(Beer's Law)
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==Beer's Law==
==Beer's Law==
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* Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine, inosine, and ADA. Their absorbance are listed on the table below.
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* Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine and inosine Their absorbance are listed on the table below.
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|''Wavelength''
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| align="center" style="background:#f0f0f0;"|''Adenosine''
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| align="center" style="background:#f0f0f0;"|''Inosine''
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|-
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| Absorbance at 235||.57||.14
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|-
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| Absorbance at 265||.4||.6
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|-
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Revision as of 11:44, 9 December 2012

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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.
Wavelength Adenosine Inosine
Absorbance at 235.57.14
Absorbance at 265.4.6


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