BCMB20005
Guidelines:
The following information must be included in your report.
Title Include a relevant, informative, yet concise title (do not include the subheading of 'title' before your title).
Aim(s) Re-write the aim(s) in your own words (consider the results when writing the aims of the experiment) for Experiment 7.
Materials and methods Outline the relevant techniques and provide a corresponding citation of the Lab Archives/Lab Manual protocol.
Use text to describe the methods, key variables that can influence the experimental outcome, controls (where applicable) and sample differences (this may include: (wavelengths, concentration ranges, reagent final concentrations, standard curve range, specific equipment, diluents & reagent blank, enzyme concentrations etc).
Where appropriate, use tables to describe the composition of samples (with the exception of the Lowry method where you should use text to describe your samples, similar to the approach used in Experiment 5’s report), being sure to refer to final concentration of reagents where appropriate (as learned in tutorials). Buffer composition for IEX, vapour diffusion method and lysozyme activity assay are integral to the outcome of the experiment and therefore should be clearly defined in your method.
Any tables included should be re-labelled/numbered to suit your report.
Results - Figures, Tables & Calculations Include the following results in the results section and remember to use concise introductory and linking text to provide flow to your results section. Note, students can present the order of results as they deem appropriate as long as it is logical.
Appropriately labelled & captioned figures
· Crystallisation of lysozyme (you are permitted to include up to 4 images of your selected panel or a minimum of 1 image)
· SDS-PAGE gel (including sample layout, identification of molecular weight standard sizes, and positions of key proteins in egg white)
· Graphs of assays of lysozyme activity for fractions A–E (including clearly labelled tangents)
· Standard curve for the estimation of protein concentration
Tables (including an appropriate table heading located in accordance with standard conventions for scientific writing)
· A table combining: Absorbance and protein amount data from the Lowry assay for the purification fractions; alongside description and volumes of each purification fraction
· Purification table including the following parameters calculated for each FRACTION (where applicable) total protein amount and concentration, total units of enzyme activity, specific activity, yield and purification factor
· Table of crystallisation results. Students can include any three of the following descriptions: number of crystals, sizes, spatial distribution of the crystals within the droplet, and/or colour intensity.
Calculations - PLEASE TYPE YOUR CALCULATIONS (updated 23/5 9am).
1. Include calculations for lysozyme activity assay for each relevant tangent (A-E)
2. Include the full workings of the calculations for the purification table using Fraction E as an example. Include: total protein concentration (in mg mL-1), total protein (mg), specific activity, yield, purification factor.
Note: Students must complete calculations for all fractions (with final values for each fraction presented in the purification table) but can simply present the full working-out for Fraction E calculations.
Discussion (Word limit: 800 words)
Consider the results of the three weeks of Experiment 7 and use the discussion points below to frame. your discussion. The discussion section should be presented as prose, not as distinct answers to the questions contained within the discussion points. DO NOT present your discussion section in bullet points.
The Discussion section must tie together the three weeks of results and must address:
1. How lysozyme was isolated, including the purpose of Buffer 2 compared to Buffer 1 in the context of ion-exchange purification.
2. How increasing the buffer pH from 9.0 to 10.4 and increasing ionic strength assists in the elution of lysozyme from the CM-Sepharose (recall that Buffer 2 was used to elute the proteins bound to CM-Sepharose-pellet) Hint: Recall and refer to fractional charge from Day 1 questions and calculate concentration of Na+ ions in buffer 1 and 2.
3. Correlation of the data generated from the three-week experiment (including SDS-PAGE, crystal results, activity data, etc) to discuss the efficacy of the purification process (in relation to yield and fold-purification).
4. Discussion of two possible methods that could be introduced and how they could be used to improve the purification of lysozyme. Hint: Consider SDS-PAGE results and think about the characteristics of any contaminating proteins.
5. A comparison of the crystallization results obtained for commercially purified lysozyme and your purified lysozyme, including discussion of the purpose of Buffer A and Buffer B in the preparation of the lysozyme crystals. Hints: Consider if crystals formed from both the commercial lysozyme and fraction E? If not, propose reasons why not? Did the concentration of protein affect the results of crystallization (number and/or size of crystals)? Relate your outcome to your actual results of protein concentration.
Note: Always refer to the corresponding figure/graph number when discussing specific results.
Conclusion Provide a concise conclusion for Experiment 7 with reference to the aims, using supporting statements of evidence.