LABS 7&8 MINI REPORT

Refer to the general guidelines for Mini Lab Reports on Canvas, found here (link opens in a new tab): Lab Mini Reports

This report is done in pairs.

Methods (Labs 7-8) 

Write a thorough Methods section for labs 7 and 8. A few general reminders:

• When mentioning buffers, include the actual recipes. Don’t just say “Buffer 1”
• Include the protein purified; if mutant, identify the mutant.
• Include the equation you used to determine protein concentration and Rf calculation and be sure to define the variables. (See note on equations on at Mini Lab Reports)
• State exactly which samples were run on the gel

Results (Labs 6-8)

• A single plot showing the growth curves for each of your three cultures.
• A description of the effect the IPTG had on cell growth for both the mutant and wild type cultures.
• Include a picture of your gel, indicating the following:
  • The identity of the sample in each lane
  • The electrode polarity (positions and charge of anode and cathode)
  • The position of the top of the resolving gel
  • The position of the dye front
  • The size of the molecular weight markers in the gel
  • Highlight and label any bands of interest (some suggestions below)
• A molecular mass calibration curve for the standards by plotting the Rf versus the log of the MW. What is the linear range of the molecular weight marker? Include the best-fit line on the graph. Describe the graph including the shape of the line and what it tells you about the size range of the markers.
• From your examination of the expression induction samples (from lab 6), identify and label on the gel any induced protein bands. Calculate Rf-values for these bands and determine the molecular weight of these proteins by comparing the Rf for this band to the molecular weight standards. Based upon your molecular weight calculation of the most highly induced protein band, did you induce a protein with the same MW as HCAII? At what time, and under what conditions, did you achieve maximum expression of this protein according to SDS-PAGE?
• For the purified HCAII samples (lab 7), describe the gel results and what they tell you about the protein purification process. Calculate the Rf value for the brightest purified band; does its MW match that of HCAII?
• The concentration of your protein after dialysis, in μM

 Discussion (Labs 6-8):

• What is the IPTG doing in the cells that would affect their growth? Was there a difference in how the two different cultures were affected by IPTG? Did you expect there to be a difference? Why?
• Which samples did you run on the gel and why did you run those samples? What information were you trying to get from those samples? Did you get the information you expected? If not, why not?
• Do you think you induced HCAII expression in E. coli in lab 6? Use your data to support your conclusion. Did you induce expression of any other proteins? If so, what were their MWs, and what might they be?
• Do you think you purified HCAII in lab 7? How well did your column work? Use your data to support your conclusion.
• If anything went wrong in your purification or gel running, describe what it was and what you would do to correct the problem if you were to repeat the purification/gel running.
  • If there was a gel issue, here is a helpful place to start with.
• Although your protein is not completely pure, it is pure enough for the rest of the experiments in this class. Describe what you would do as a next purification step if you needed very pure protein (e.g.. pure enough for X-ray crystallography or NMR).
• Briefly describe what you will do next with your purified HCAII.