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25 Aug
2019

Biochemical fractionation of milk proteins | Good Grade Guarantee!

ii)iii) orksheet B (covers practical 2)

Name:

Prac Partners:

Prac date:

Due date:

1) Electronic Format
The worksheet must be produced entirely in an electronic fashion, however, we do not accept ePens. Hand-written or hand-drawn components, be it as original or as scanned image, will not be accepted/not receive any marks! Submit electronically as either a Word or PDF file via the designated CloudDeakin dropbox.
2) Plagiarism and Collusion
Before submitting your worksheet to the dropbox submit it to the Check Your Work: Turnitin Assignment Folder available on CloudDeakin. In case that we find that work is the result of plagiarism (from foreign source like internet or fellow student) or collusion we report this to the Faculty Academic Progress and Discipline Committee.
Note: It is NOT sufficient to submit your worksheet only to the Turnitin Folder! Your worksheet will be marked only if you submit it to the appropriate Dropbox!
3) Due dates for worksheet submission:
Worksheet A is due 5.00 pm two teaching weeks after practical session 1, this is the day BEFORE your prac 2
Worksheet B is due 5.00 pm two teaching weeks after practical session 2, this is the day BEFORE your prac 3
Worksheet C is due 5.00 pm two teaching weeks after practical session 4 (!), this is the day BEFORE your prac 5
Worksheet D is due 5.00 pm ONE (!) teaching week after practical session 5 (!)
4) Referencing
All foreign sources of information, i.e. information that was not provided in this unit, must be cited. The citation style to be used is Harvard style, for more information about this style and referencing in general see: https://www.deakin.edu.au/students/studying/study-support/referencing. Citations will be considered only when they are placed as in-text citations such that it is clear which statement they do support. Furthermore, bibliographic details of a citation must be listed within the question where they are used, reference lists at the end of the worksheet will not be accepted. The citation needs to be a quality journal article, book or refereed website. No marks are awarded if Wikipedia or poor-quality websites (eg. the website of a course at another institution) are cited.
The practical manual, the prac script, OLMs and lecture notes can NOT be cited. Citations of text books are often most appropriate for the level of this unit and therefore encouraged.If you do cite a textbook you MUST indicate relevant page numbers.
5) Learning Outcomes:
Q1, Q2a: Written communication. Presentation of experimental data.
Q3: Quantification skills. Application of a mathematical equation to solve a typical problem encountered in the biochemistry lab.
Q2b,c, Q4:Critical thinking & problem solving. Use of logic and discipline specific knowledge to describe and predict an experimental outcome
1a. Produce a figure using the image of the SDS-PAGE gel that you created in the prac
(1.5 marks)
Only the lane for the molecular mass standards and the skim milk sample lane are to be presented. You will be given 0 marks for Q1a if other or additional lanes are presented! You can cut and paste the two required lanes out of the original gel image file.
Explanatory note: The teaching team strictly enforces the above indicated requirements for presentation of SDS PAGE data to ensure the originality of 2019 worksheet submissions.
Indicate the identity of the lanes (what has been loaded in which lane), the origin of electrophoresis (ie the bottom of the well into which the protein was loaded), electrophoresis front (the most advanced part of the electrophoresis in the gel), the molecular mass of markers proteins and indicate the casein bands and heat resistant low molecular mass proteins. You can use e.g. Power-Point, Photoshop or Illustrator or similar software for his task.
Write a concise figure legend with a minimal number of words that allows the reader to understand what they see. Note: A figure legend gives some experimental information to provide context but does not reproduce the entire procedure in detail. It also defines any terms or symbols or describes equation terms to allow the reader to understand the figure by itself. A figure legend should not contain any interpretation of the results!
1b) Provide a concise description of the proteins that you observe via SDS PAGE of skim milk.
Describe the proteins in terms of their size and abundance.
Provide a conclusion of how your SDS PAGE analysis of milk fractions allows you to confidently identify casein proteins that are present in the milk sample.
Provide a conclusion of how your SDS PAGE analysis of milk fractions allows you to confidently identify small molecular mass proteins that are present in the milk sample.
Must be less than 100 words total (1.5 marks)
i)
ii)
iii)
2a. Create a standard curve using molecular mass standards resolved in your SDS PAGE. To do this, draw a graph of the log molecular mass of the different protein standards on the y-axis against the migration distance of the protein standards within the polyacrylamide gel on the x-axis. See figure 13 on page 97 of your text for an example. Using Excel, create a scatter plot (data points that are not connected) and generate a linear regression and straight-line equation. Note that the slope of the graph should be negative (The y values (log MW) decrease as the x values (Distance migrated) increase). Express the equation in terms of log molecular mass and distance migrated. Give the graph a title and write a figure legend, which would explain the graph. The figure legend should allow the reader to understand the graph. (1 mark)
The colour of the x-axis and y-axis and the labelling of the axesof the graph MUST be BLUE(you will receive 0 marks for question 2a in case that a different colour is used).
Explanatory note: The teaching team strictly enforces the above indicated color requirements in order to ensure the originality of 2019 worksheet submissions.

2b. Using the equation of the straight line generated from the above standard curve, calculate how far an 80,000 Da protein would migrate in the SDS-PAGE gel. Show your complete working for the calculations including units to the nearest whole millimetre. State your conclusion in a complete sentence (1 marks)
2c. You are studying a protein and used SDS-PAGE to estimate the molecular mass. On the first attempt, you estimated that the size of the protein was 60 kDa. On the second attempt you estimated that the size of the protein was 30 kDa. Going through your notes, you discovered that in the first attempt, you forgot to add the 5% (v/v) ß-mercapto ethanol to the sample buffer. As per prac 2, the sample buffer did contain 4% (v/v) SDS and was heated 95oC for 4 minutes. On the second attempt the sample was prepared by mixing it with a buffer containing 4% (v/v) SDS and 5% (v/v) β-mercapto ethanol and it was heated at 95oC for 4 minutes.
Explain:
Why the protein size estimate was different between the first and second SDS-PAGE attempt.
Include a description of the function of the ß-mercaptoethanol in the system.
Provide a conclusion about the structure of the studied protein.
Must be less than 100 words total (1.0 mark)
i)
ii)
iii)
3a. Draw a graph of absorbance against fraction number for the gel filtration chromatography (2.0 marks).
Using Excel you will need to create a single graph (Straight marked scatter plot) with the fraction number on the x-axis and two y-axes (one y-axis on the left for the absorbance of haemoglobin (Abs 400 nm) and one y-axis on the right for the absorbance of vitamin B12 (Abs 550 nm). Provide separate plots for the absorbance at 400 nm and the absorbance at 550 nm. You will need to adjust the scale on the individual y-axis such that both graphs show comparably sized peaks (If you would use a single y-axis, then the values of one data set would be 10 -100 times larger compared to the other one resulting in a very small peak for the smaller values; this is why you use two y-axes).
The colour of the x-axis and y-axes and the labelling of the axesof the graph MUST be BLUE(you will receive 0 marks for question 3a in case that a different colour is used).
Explanatory note: The teaching team strictly enforces the above indicated color requirements in order to ensure the originality of 2019 worksheet submissions.
Give the graph an appropriate and descriptive title and label the axes, including appropriate units. Provide a figure legend, which concisely describes the graph that is shown and the experiment that underlies the measurements. Note:A figure legend gives some experimental information to provide context but does not reproduce the entire procedure in detail. It also defines any terms or symbols or describes equation terms to allow the reader to understand the figure by itself. A figure legend should not contain any interpretation of the results.
3b. A SLE222 student found that in the P60 gel filtration chromatography, there were two absorption peaks at 400 nm, the first peak occurred in fraction 2 while the second occurred in fraction 9. They reasoned that the first was haemoglobin and that the second was vitamin B12. They concluded that gel filtration chromatography separates molecules on the basis of their mass. Haemoglobin had a molecular mass of 63 kDa while vitamin B12 had a molecular mass of 1.4 kDa. Haemoglobin was eluted first since it was heavier than vitamin B12 and thus due to gravity sunk to the bottom of the mobile phase as it moved through the column.
Did the student draw the correct conclusion from the chromatography result? Justify your answer.
Describe the elution profile of your experiment including a description of the theory behind the separation of molecules by gel filtration chromatography.
Must be less than 150 words total (1.0 mark)
i)
ii)

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