Posted: September 16th, 2016

Accurately assess the amount of protein extracted from a sample of bacterial cells

Assessment item 4: Assessment of practical skills. Intent: While one could have a career in science that involves analysing data produced by others, the real fun is performing your own experiments. But experiments only have value if the data generated is reliable which means that you have to be able to perform the methods properly. The Proteomics subject has a great emphasis on practical skills and thus you have to demonstrate that you understand the methods you are using and that you can perform them with high accuracy and precision. The three tasks that make up this assessment let you demonstrate that you can read instructions and perform some fundamental proteomics techniques. Objectives: For you to demonstrate the laboratory skills necessary to undertake proteomics based research. Proteomics Practical Manual Page 9 This assessment task contributes to the development of the following graduate attributes: 1. Disciplinary knowledge and its appropriate application 3. Professional skills and their appropriate application Task: Three tasks will be performed by you. 1) Accurately assess the amount of protein extracted from a sample of bacterial cells. The task will demonstrate that you can follow the instructions within this manual for the extraction of protein, the performing of a protein assay with dilution series of a known standard, the plotting of a standard graph and the determination of the concentration and amount of the extracted protein. Assessment criteria: A standard graph with an R2 value of 0.9 or greater. A certain total amount extracted protein from the sample for use in further experiments. 2) Cast a polyacrylamide gel and use it to separate their previously isolated protein sample by SDS-PAGE. The you will then fix the gel and stain the proteins with Coomassie Blue stain. Assessment criteria: Production of a correctly set and useful polyacrylamide gel. Following electrophoresis, gel staining and destaining, the gel will be inspected for correct protein loading and protein band resolution. 3) Perform an in-gel trypsin digest on an isolated protein spot from 2D-PAGE, recover and desalt the peptides using a C18 ZipTip, load the peptides onto a MALDI target and perform tandem MS. Assessment criteria: MALDI-TOF MS and MSMS spectra showing good peptide recovery, the presence of auto-catalytic trypsin fragments and high quality database match with Mascot facilitated database search.