User:Eleni N. Kalivas/Notebook/CHEM-571/2013/09/10

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Objective

The objective for today was to ascertain the concentration of Adenosine Deaminase via analysis of the UV-vis spectra. Since protein concentrations are difficult to measure Bradford Assay, which is a protein binding dye, will be used. The specific dye used is called Coomassie Blue. Once the dye binds with the protein it undergoes absorption changes. We will be making a calibration curve using the Bradford Assay data for the protein.

Adenosine Deaminase

The protocol called for 10mg of protein in 2mL of solution. Due to insufficient protein we were only about to use 3.6mg per 2mL which had a final concentration of 1,800 μg/mL. This was then used to create six standard solutions:

  • table for 6 standard solutions

Adenosine Deaminase (6) standard Table.png

Tables zem-enk.png

Absorbance Spectra of Adenosine Deaminase

  • Trial 1: Absorbance of Adenosine Deaminase:

AD absorbance trial -1 zem-enk.png

The graph includes a key where starting at number 1 (our highest concentration), until 6 (which is our lowest).


  • Trial 2: Absorbance of Adenosine Deaminase:

AD trial-2 zem enk.png


  • Trial 3: Absorbance of Adenosine Deaminase:

AD trial-3 zem enk.png

  • Results: based off the the two trials run, the absorbance spectra were clearly flawed. Therefore after further analyzation we decided to discard all dilutions, and to re make new dilutions based off of the same stock solution with the same dilution values, in order to retest to retest the adenosine deaminase and create a better spectra that will provide a calibration curve. These new diutions will be prepared and tested tomorrow.

Atomic Absorption Standards Prep

  • Gold nanoparticle (AuNP) standard solutions were prepared to be be tested tomorrow.

The following standard molarities were prepared:

Standard Dilutions Table enk zem.png

The AuNP dilutions were from a stock solution of 1000 (±) 10 μL/mL, 10% HCl: Gold Nanoparticles Standard.png