User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/31

Separate Protein and Lysozyme UV-Vis

Goals

• Separate the proteins from the cells
• Run UV-Vis on the AuNP/Lysozyme solutions

Procedure

• The same procedure from 2012/09/25 was followed in order to separate out the proteins from the cell organelles.
  • The cells were shocked open by alternating sonication for 30 seconds followed by 30 seconds on ice.
  • Afterwards, the cells were centrifuged at 18000 rpm and 4°C for 2 hours.
  • The supernatant was collected and purified through vacuum filtration with filter pape membrane in which the holes measure 450nm. The filtered product was collected and stored in the refrigerator for approximately 1 week.

• For information regarding the AuNP lysozyme UV-Vis spectras please see Michael's Notebook. These spectras were taken using the same samples as were made last week

Data

Conclusions

• The separation process ran smoothly except for during the vacuum filtration. There seemed to be a defect in the clamp being used which caused some of the supernatant collected to leak out. A glass pipette was used to recollect as much as possible and placed back through the filter. This defect may be the cause for any loss in ADA collected when conducting the FPLC.
• Samples 134-140 showed a variation in the baseline and as a result, in order to determine whether this was due to lysozyme or a variation int he cuvettes, these four trials were repeated using the same quartz cuvet. A blank of water was run using the same cuvette and subtracted from each spectra manually.