Phosphoenolpyruvate Carboxylase - Practical Report Writing - Nursing Assignment Help

Assignment Task

Practical Report

Introduction: 
Include a brief background about Phosphoenolpyruvate Carboxylase (PEPC), what role it may have in biological systems, lead onto the aims of the practical.

Result: 
Purification
• This is a guide; you can include more in your result section if you wish.
• Explain, the procedure that was used to purify PEPC (all steps).
• Find the pI of PEPC, and explain why DEAE-cellulose was chosen as the chromatography
media.
• What steps were taken to preserve PEPC activity during the procedure?
• Explain the assay that you used to detect PEPC. Was there any activity without PEP? What
does this tell you? Is there any PEPC activity in S2? Why would there be no activity?
Include:
Figure 1. Ion-exchange profile for PEPC purification. X-axis, fraction number, Y-axes PEPC activity andOD 280nm, you will need to write a legend.
Table 1 Purification Summary Balance sheet
Figure 2. SDS-PAGE
• Print the photograph of your gel
• Label the printed photograph of your gel with the lane numbers, i.e., 2, 3, 4 etc.
• Label the standard proteins with their molecular weights in kD.
• Write a legend for the gel in which you describe the contents of each lane (this must include the
amount of protein loaded per lane in µg).
• Determine the Rf values for each of the standard proteins. Plot the Rf values against the log molecular
weight of the proteins to obtain a standard curve. Fit a straight line to the points using linear
regression. Put this in your Appendix (see below)
• Use this plot to estimate the molecular weights of the prominent protein bands in your gel.
Figure 3 - Western Blot
• the Western Blot labelled in the same way as the SDS-PAGE gel (see session 4)
• a standard curve for the standard proteins on the Western Blot (see session 4)
• a table showing the molecular weights of the protein bands of interest on the Western Blot
2.2 Enzyme Inhibitor kinetics
Figure 4 – Plot of Reaction rate (v) vs PEP concentration – three curves for each Malate concentration –include Figure legend
Figure 5 – Double reciprocal (Lineweaver-Burke) plot – used to assess what type of inhibitor malate is.1/v vs 1/PEP
Table 2 – Table of Km and Vmax at each malate concentration, (anyone who submits a correct Kicalculation for malate will receive a bonus 1% (above the 25% allocation) for this assignment, but youneed to show me in appendix how you calculated it).

Discussion:
Discuss your purification protocol; did it work what were yield and the enrichment? Could it be improved? How does it compare to protocols in the literature?
• For SDS-PAGE, which of these bands is likely to be the PEPC subunit? Why?
• For SDS-PAGE, compare what you see in the different lanes of the gel. Do some of the protein bandsdisappear and others become enriched during the purification protocol? Does the band that yoususpect to be the PEPC subunit become more prominent? Why?
• a discussion of the conclusions which you can draw from the results of your Western Blot
• What type of inhibitor is malate? Is this relevant in the physiology of a cell? Bring in anything youlearnt from your CML exercise.
• You should finish with some conclusions about the purification protocol and the inhibition studies.

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