Sandbox3H04: Difference between revisions

The structural viewing program Chimera was used to view the specific amino acids from SPRITE in the theorized functional group. 1a8q was the primary match used to model matching H304’s 104 serine at 1a8q’s 94, 220 aspartate at 223, and 248 histidine at 252 respectively with a RMSD of 0.452. An overlap was performed to view the functional alignment which can be viewed here.[[Image:ChimeraAlignmentBS.png |300px|center|thumb|]]

 

Protein Purification was the first step in getting to a point of experimental data. The protein was grown in E. coli solution that had been tagged with an antibiotic resistance to isolate the protein. The pellet was then lysed with a buffer and sonicated to break up the sample. The resulting sample was centrifuged and the liquid was collected and placed into a separatory column to gather elutions. All steps had portions that were taken throughout to view where the protein was best concentrated. The first experimental occurred from a Bradford assay of known standards to determine which elution had the highest concentration of protein. (Graph to show elution 1 had highest amount). The second experiment was running the portions through all steps on an SDS-PAGE gel to view the purify and amount of protein. It was determined that the protein had expelled through the wash before the elutions were collected. (Include sds-page here) The wash was used to test enzymatic activity by adding PNPP, a buffer, and protein, then recording the absorbance for around 30 minutes. Testing showed a sharp increase in absorbance from 22 to 32 minutes meaning PNPP was cleaved to produce the increase in intensity. The experiment was run again at body temperature using a well plate reader with similar results but much higher increase in intensity.