Reference no: EM132869284

Biochemistry Teaching Laboratory

Learning Objective 1: To appreciate that separation of biological macromolecules is necessary for research and industry;
Learning Objective 2: Be able to interpret a column chromatogram;
Learning Objective 3: Be able to interpret an electrophoretic gel;
Learning Objective 4: Recall that Sephadex is a size exclusion resin, and that there are various mechanisms that enable separation in column chromatography (anion exchange, solubility gradient);
Learning Objective 5: Recall the differences between agarose and polyacrylamide gel electrophoresis.

Preparation
You can read more about chromatography and electrophoresis in the appendix to chapter 5 in your text, Garret & Grisham. Complete the pre-lab quiz on LearnJCU before attending the practical.

Exercise 1

Figure 1, below, shows a sample of a protein mixture which has been run on a Sephadex column at acidic pH. Recall that Sephadex is a size exclusion resin, and that the larger molecules elute more quickly because they do not fit into the pores of the Sephadex beads.

1. Determine the fraction # (mL) at which each protein elutes by measuring from the top of the peak to the x-axis (each fraction contains 1 ml). Use the data to complete Table 1 below.

2. Plot the log10MW of the protein standards against the fraction number on the graph paper provided.

3. Extrapolate from the graph the molecular weights of the unknown proteins in the mixture

Q1. a) What are the molecular weights of the three proteins, and
b) Which fraction would contain cytochrome c? (See your textbook)

Q2. From what type of tissue would you choose to isolate cytochrome c?

Q3.What kind of purification and/or enrichment steps would you take (other than chromatography) to maximize the yield of cytochrome c from your tissue?

Exercise 2

Objective - students look at data from a column and a gel and deduce the size of the protein and the number of sub-units joined by di-sulphide bridges.

The unknown protein was run on the same Sephadex column under the same conditions as that in Figure 1. The elution profile for this protein is shown in Figure 2. Calculate the weight of this protein in the same way as you did you exercise 1 (is this the weight of the native protein or the subunits?).

Then, utilizing the figure of the denaturing SDS-PAGE gel (Figure 3) calculate the size of the 2 bands. To do this you will have to construct a standard curve based upon the molecular weight markers in lane 1 and the distance traveled of each band. Measure from the top of the gel and plot the log(molecular weight) vs distance travelled. Show your standard curve including title and axis labels.

Q4. Why is your curve not a straight line? Which part of the curve should you use to determine your protein size?

Q5 Using the information from the Sephadex column and the SDS-PAGE, suggest how many of each subunit are in this protein, giving a short justification.

Exercise 3

Objective - Determine the subunit weight(s) of an unknown protein using SDS-PAGE electrophoresis and gain experience loading an SDS-PAGE gel.

1. During class each person will be asked to load one of four different protein samples onto an SDS-PAGE gel (please note the sample ID of the protein you load, the gel ID and the lane number which you load you sample into), a marker will also be loaded. The gel will then be run for 1.5 h, stained using Coomassie and then destained the following day.

2. Images of the gels will be posted on LearnJCU prior to the report due date. Measure the distance travelled by the proteins in the ladder and your unknown. Using this information, construct a standard curve and determine the size of the subunit(s) in your protein. You will be required to provide the standard curve and a statement saying what the size of the subunits were.

Attachment:- Biochemistry Teaching Laboratory.rar