Lab 8: MINI LAB REPORT

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

Methods (just lab 8)

• State exactly which samples were run on the gel
• Be sure to include any equations you used to calculate Rf values

Results

• Include a picture of your gels, 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: 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: 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:

• Which samples were run on the gel and why? What information would you expect to get from those samples? Did you get the information you expected? If not, why not?

• Do you think HCAII expression was induced in E. coli? 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 HCAII protein was purified? How well did the purification column work? Use data to support your conclusion.

• If anything appears to have gone wrong in the purification, describe what is was based on the gel, and how you might be able to correct the problem if you were to repeat the purification.

• 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 the purified HCAII.