User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/04/17

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Objective

Determine the protein concentration of the MBP-intein and GFP proteins. Also, run intein chemistry reactions overnight.

Description

UV-Vis of Proteins

  1. Place 10x dilute MBP-intein in the UV-Vis, and take a spectrum from 200nm to 800nm.
  2. Repeat with the mutated GFP protein.
  3. Analyze both spectrums to determine concentration.

Intein Reactions

Tube Number 1 2 3 4 5 6 7 8 9 10 11 12
Amount of MBP-intein 250 μL 250 μL 250 μL 250 μL 250 μL 250 μL 0 μL 0 μL 0 μL 0 μL 0 μL 0 μL
Amount of MBP-intein-gold nanoparticle 0 μL 0 μL 0 μL 0 μL 0 μL 0 μL 250 μL 250 μL 250 μL 250 μL 250 μL 250 μL
Amount of GFP 250 μL 500 μL 750 μL 250 μL 500 μL 750 μL 250 μL 500 μL 750 μL 250 μL 500 μL 750 μL
Amount of Buffer (50 mM Tris-HCl, pH 8.0 and 1 mM CaCl2) 500 μL 250 μL 0 μL 500 μL 250 μL 0 μL 500 μL 250 μL 0 μL 500 μL 250 μL 0 μL
Temperature (°C) 25 25 25 25 25 25 37 37 37 37 37 37

Data

The corrected absorbance of the mutated GFP protein at 485nm is 0.033.

A=εbc, where A485=0.033, b=1cm, and ε=61000cm-1M-1 The diluted concentration is calculated as 0.54μM. The concentration of the stock GFP is 5.4μM.

The corrected absorbance of the MBP-intein protein at 280nm is 0.02.

A=εbc, where A280=0.033, b=1cm, and ε=77600cm-1M-1 The diluted concentration is calculated as 0.26μM. The concentration of the stock MBP-intein is 2.6μM.




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