User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/09/13

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Objective

1. Determine the unknown concentration of maltose-binding protein (MBP) using BSA standards and a Bradford assay.

2. Prepare for and start the growth of E. coli for protein expression.

Description

  1. Standards were set-up in the following way in plastic cuvettes:
Standard Amount of Bradford Dye Amount of 14.6μg/mL BSA Amount of Distilled Water Final Concentration in Cuvette
Blank 200 μL 0 μL 800 μL 0 μg/mL
Standard 1 200 μL 50 μL 750 μL 0.73 μg/mL
Standard 2 200 μL 100 μL 700 μL 1.46 μg/mL
Standard 3 200μL 250μL 550μL 3.65μg/mL
Standard 4 200μL 400μL 400μL 5.84μg/mL
Standard 5 200μL 550μL 250μL 8.03μg/mL
Standard 6 200μL 700μL 100μL 10.22μg/mL
  1. Spectra from 200nm-800nm was taken of each of the standards.
  2. The wavelength recorded at 595nm was used to create a standard curve for protein concentration.
  3. The MBP protein at its unknown concentration was diluted by a factor of 10 by combining 100μL of the MBP with distilled water.
  4. 10μL of this more dilute protein was then mixed with 790μL of distilled water and 200μL of Bradford dye in a plastic cuvette.
  5. A spectrum of this mixture was taken from 200nm-800nm.
  6. A spectrum was also taken of 10μL of the 10x dilute MBP with 990μL of distilled water in a quartz cuvette.

Preparation for Expression

  • Preparation of 1L of Rich Media: 10g tryptone + 5g yeast extract + 5g NaCl + 2g glucose
  • 25mL of sterile rich media was added to each of four 250mL Erlenmeyer flasks that had been previously autoclaved. 25μL of 100mg/mL ampicillin (sterile-filtered) to each flask. Each flask was then inoculated with a bit of E.coli that was scraped out of a glycerol stock. The E.coli was ER2566 with the pMAL-pIII plasmid. These flasks were grown overnight at 37°C at 250rpm.
  • 0.1M IPTG was made with 0.095g of IPTG and 4mL of distilled water. Once the IPTG was dissolved the solution was sterile-filtered.
  • 100mg/mL ampicillin was made with 0.5g of ampicillin and 5mL of distilled water. Once the ampicillin was dissolved the solution was sterile-filtered.

Data

Here is the standard curve that was created by plotting the concentration of BSA in each standard against the corrected absorbance for that standard at 595nm:


  • The equation of the line was determined by Excel to be y=0.0014x
  • The absorbance of MBP at 595nm is 0.665. The corrected absorbance for this protein is 0.665-0.65, which is 0.015.
  • The concentration of the protein in the plastic cuvette is 10.714nM. (0.015/0.0014=10.714)
  • Since the protein was diluted 1000 times in this cuvette, the concentration of the undilute MBP is 10714nM or 10.714μM.

The 200nm-800nm spectrum that was taken in the quartz cuvette without Bradford dye:

Using the absorbances from this spectrum and the now known concentration of the MBP in the cuvette (1000x dilute) the molar absorbtivity can be determined for all wavelengths using the equation A=εbc, where A is the absorbance, ε is the molar absorbtivity, b is the pathlength (1cm), and c is the concentration. A graph of the wavelength vs. the molar absorbtivity was created:

Your molar absorptivity is incorrect. Check your calculations. You may also want to correct your data so that the baseline goes to zero -- but make proper note of doing so. Matt Hartings 20:21, 20 September 2011 (EDT)

Notes



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