User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/08

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Results from Yesterday

"Protein Gel"

Although this is not the best picture of the gel, it is also not a great gel. The protein was not able to be resolved on this gel (the ladder did not separate and is barely visible in the first lane). The protein in lanes 3, 5, and 7, which have Tubes 1-3 of the triple mutant, respectively, did not look like they traveled much. So, although I finally got a gel to solidify, it unfortunately did not tell us anything about the protein.

Photo (12).JPG


Transformations

Nothing grew once again. Dr. Hartings will do a transformation on Monday, since I haven't had any luck with them for the past couple weeks.

Effect of Temperature on Asc Hb Triple Mutant Spectra

  1. The 6-cell unit temperature was lowered to 0.5°C.
  2. 984μL of buffer was mixed with 16μL of the concentrated Asc Hb triple mutant (M8S/M33S/M103S) from Tube 1 (1.2mM) in a quartz cuvette.
    • Final concentration in the cuvette was 19.2μM.
  3. The cuvette was placed in the spectrophotometer and given time to cool.
    • There was a problem with "fogging" on the sides of the cuvette with the lower temperatures, so the cuvette was removed and wiped with a Kimwipe before the readings at lower temperatures were taken.
  4. A spectrum from 800-200nm was taken of the protein in buffer.
  5. The temperature was increased to 10°C and a spectrum was taken again.
  6. This was repeated at 20°C, 25°C, 30°C, 40°C, 50°C, 60°C, and 70°C.
  7. A spectrum from 800-200nm was taken of just the buffer (25mM Tris, 50mM NaCl, pH 8) at 25°C to be used as a baseline.

Results

The absorbances of the buffer were subtracted from the absorbances that were collected at the various temperatures with protein. The corrected spectra were plotted in Excel:

Temp effects triple mutant 060812.JPG

Here is the same Excel graph, but instead zoomed in to the peak just after 400nm:

Triple mutant temperature effects zoomed060812.JPG

Effect of Urea on Asc Hb Triple Mutant Spectra

  • 5M Urea was prepared today by dissolving 3.00g of Urea in dH2O. The final volume of the solution was brought to 10mL with dH2O in a volumetric flask.
  • All of these spectra readings were taken at 25°C.
  1. A spectrum from 800-200nm was taken of the buffer (25mM Tris, 50mM NaCl, pH 8) to be used as a baseline.
  2. Spectra of the following mixtures were taken from 200-800nm. For each mixture, the protein was added last and pipetted up and down into the mixture. The cuvette was then covered with parafilm and inverted a few times to thoroughly mix.
Mixtures with Various Concentrations of Urea
Amount of Buffer Amount of 5M Urea Final Concentration of Urea in Cuvette Amount of 1.2mM Triple Mutant Asc Hb
984 μL 0 μL 0M 16 μL
964 μL 20 μL 0.1M 16 μL
934 μL 50 μL 0.25M 16 μL
884 μL 100 μL 0.5M 16 μL
834 μL 150 μL 0.75M 16 μL
784 μL 200 μL 1M 16 μL
684 μL 300 μL 1.5M 16 μL
584 μL 400 μL 2M 16 μL
484 μL 500 μL 2.5M 16 μL
384 μL 600 μL 3M 16 μL

Results

The absorbances of the buffer were subtracted from the absorbances that were collected at the various concentrations of urea with protein. The corrected spectra were plotted in Excel:

Urea effects riple mutant 060812.JPG

Here is the same Excel graph, but instead zoomed in to the peak just after 400nm:

Urea effects riple mutant 060812 zoomed.JPG

Effect of Guanidine-HCl on Asc Hb Triple Mutant Spectra

  • 5M Guanidine-HCl was prepared today by dissolving 4.78g of Urea in dH2O. The final volume of the solution was brought to 10mL with dH2O in a volumetric flask.
  • All of these spectra readings were taken at 25°C.
  1. A spectrum from 800-200nm was taken of the buffer (25mM Tris, 50mM NaCl, pH 8) to be used as a baseline.
  2. Spectra of the following mixtures were taken from 200-800nm. For each mixture, the protein was added last and pipetted up and down into the mixture. The cuvette was then covered with parafilm and inverted a few times to thoroughly mix.
Mixtures with Various Concentrations of Guanidine-HCl
Amount of Buffer Amount of 5M Guanidine-HCl Final Concentration of Guanidine-HCl in Cuvette Amount of 1.2mM Triple Mutant Asc Hb
984 μL 0 μL 0M 16 μL
964 μL 20 μL 0.1M 16 μL
934 μL 50 μL 0.25M 16 μL
884 μL 100 μL 0.5M 16 μL
834 μL 150 μL 0.75M 16 μL
784 μL 200 μL 1M 16 μL
684 μL 300 μL 1.5M 16 μL
584 μL 400 μL 2M 16 μL
484 μL 500 μL 2.5M 16 μL
384 μL 600 μL 3M 16 μL

Results

The absorbances of the buffer were subtracted from the absorbances that were collected at the various concentrations of Guanidine-HCl with protein. The corrected spectra were plotted in Excel:

Guanidine effects triple mutant 060812.JPG

Here is the same Excel graph, but instead zoomed in to the peak just after 400nm:

Guanidine effects triple mutant zoomed 060812.JPG