Analysis of metabolic components II
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Please note: The report must be produced entirely electronic! Hand-written or hand-drawn components, be it as original or as scanned image, will not be accepted!
Analysis of practical results
- Produce a figure with an appropriate figure legend of your SDS-PAGE gel that could be published in a scientific journal.
- Indicate all relevant information, e.g. origin, front, size of the molecular mass markers and casein and whey protein bands. 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 he sees. Note: Give some information about the methods but do not repeat all details in the legend!
- Fill in the table to report your calculated estimates for the molecular masses (kDa) of the proteins in the milk sample. You can enter up to 3 different significant bands that you observed in the respective fractions.
| Protein fraction | Estimated molecular mass (kDa) | ||
| Casein proteins | |||
| Whey proteins | |||
| Small molecular mass proteins | |||
- Draw a graph of absorbance against fraction number for the gel filtration chromatography. Using Excel you will need to create a single graph (smooth or straight marked scatter plot) with 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). You need to adjust the scale on the individual y-axis such that both graphs show reasonably large 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-axis).
Questions
4a. Explain in molecular terms the observed movement of the various sized proteins through the gel filtration column
4b. During the preparation of the samples for SDS-PAGE you mixed the protein sample with loading buffer containing sodium dodecyl sulphate (SDS) and beta-mercapto ethanol, and heated the sample. What did this process do to the sample?
- A cell consists of thousands of individual molecular components. To understand biochemical pathways it is often necessary to separate, purify and analyse the molecular constituents of a cell. Name two different biochemical/biophysical properties in which biological molecules differ from each other and think about a corresponding technique that can be exploited to separate and/or characterize such metabolites.
| Biochemical/Biophysical property | Technique that exploits this property |
| Example given: different ionic charge | Example given: separation on gel in electric field |
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- Consider an individual who had just rushed for 1 km and barely caught the desired tram before it departed. Indicate the likely concentration of metabolites/activity of enzymes that molecular analysis would reveal for this person. Increased, decreased and unchanged are describing levels relative to a healthy resting state.
| Metabolite (tissue) | Increased, decreased or stable | Reason |
| Lactate (muscle) |
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| Phosphofructokinase 1 (muscle) |
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| Glucose (blood) |
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| Glucose-6-phosphate (muscle) |
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| EtOH (blood) |
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| Glyceraldehyde-3-phosphate (muscle) |
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| ADP (muscle) |
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| Ephinephrine (blood) |
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| NADH (muscle) |
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| Fructose 1,6 bisphosphatase (liver |
