User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/25

Separate Proteins

Goals

• Separate the proteins from the cells

Procedure

• Separate Proteins

1. Remove the cells from the -80°C Freezer and place in a water bath to unfreeze the cells and bring them to room temperature.
2. Sonicate the cells using sonication instrumentation. place the sonication device inside each of vials containing the cells for 30 seconds followed placing the vial in an ice bath for 30 seconds. repeat this sequence three times. This will shock and open up the cells.
3. centrifuge the cells at 18000 rpm and 4°C for 2 hours. This will separate the organelles of the cells from the proteins we want.
4. Meanwhile, filter the binding buffer and elution buffer in order to purify them using a vacuum system of filtration and filter paper with a membrane filter in which the holes measure 450nm.
5. Once the centrifuging is completed. Use the same vacuum filter system to separate the proteins from everything else in the vials (organelles, buffer, etc.)
6. The protein was collected in falcon vials and refrigerated.

• UV-Vis

1. New concentrations of the Tris buffer were added to the Gold/BSA solution. These solutions did not express any fibers, it was just purple solution. The UV-Vis was run twice on the samples with an hour in between each run.

• Abigail E. Miller 10:56, 7 October 2012 (EDT):be careful. this sounds more like what you will do, not what you actually did. having both in your notebook is fine, but having the first only is not. where are the details? did you use two different tubes for the flask 1 cells versus the other flasks? for example: the binding buffer ( 50 mM Tris, 100 mM NaCl and 50mM imidizole, pH 7.4)(create a link to the page it was made on) was filtered by vacuum filtration using filter paper with 450 nm pore size for 2 minutes.

DATA/Calculations

• Uv-Vis:

to make a 25mL stock solution of the tris buffer at a pH of 10 1mol/L x .025= .025 mol .025 mol x 121.14 g/mol = 3.0285 g of tris

• Abigail E. Miller 11:01, 7 October 2012 (EDT):you need to note that this is a 1.0 M Tris stock.

To get the necessary concentrations of tris into the gold/BSA solutions, the M₁V₁=M₂V₂ formula was used to calculate the volume of tris needed. From there, the value of the volume of tris was subtracted from 1 in order to determine the amount of water that would be added to make the desired concentration.

Graphs of run 1 and 2

link for excel Image:TrisvaryingmolarUVVis.xlsx

• Abigail E. Miller 11:02, 7 October 2012 (EDT):what is hte difference between trials one and two? what am i looking for?

Conclusions

• Todays steps were necessary in order to separate out the proteins from the cells and collect them.
• sample 1 cells are still being tested separately due to the original discrepancy. filtration, sonication, all were performed separately for the main sample and sample 1.
• In repeating the same experiment as the previous class using varying concentrations of the tris buffer with the AuNP/BSa solution, no difference could be seen between the trials except for 5mM in the second trial. Either another trial needs to be run at this concentration, or a blank needs to be run to see if it is accountable in any way for the jump in absorbance.