Module 5: Protein Separation via Size Exclusion Column Chromatography

REPORT

Include in the following order:

- Results (raw data, table, plots)

- Answers to Questions

- Summary (objective, at least one thing that you learned, and what advice you would offer to another student doing the experiment)

**Please be aware that the estimated grade allocations for each question has been offered as a guide and may be subject to change.**

Results:

Your results section should include…

A. (3 marks) Size Exclusion Chromatography (SEC):

1.    Clear, readable photo of SEC Results with column dimensions (including any calculations if required) signed by a TA

2.    Plot a SEC Chromatogram of your results by plotting the Abs on the y-axis and fraction (or tube)      number on the x-axis. On this one graph plot both the Abs280  and Abs410 values for each tube. Use a Scatter plot with a smoothed line to represent the absorbance trend seen at each wavelength.        Include the following:

-     a proper title and axis labels/titles with appropriate units

-     a legend indicating which line for which wavelength

-     text box within the plot indicating which fractions had colour and which were positive for catalase activity

-     underneath the plot, a statement that informs the reader of the fractions that were further

analyzed by SDS-PAGE and Bradford Assay.

**Be sure to follow the graphing guidelines provided on LEARN "Graphing Data.pdf".

B. (7 marks) SDS-PAGE:

1.    Include a copy of your SDS-PAGE

- annotate electrodes

- include a legend – clarify what each lane contains with reference to the fraction number)

- assign and label the bands observed in the MW standards lane.

A list of the proteins present in the standard mix is presented below.

Q4. (2 marks) Research the properties of Myoglobin:

Go to theUniProtdatabase and search for “myoglobin AND horse”. Click on the Search button.

•    Click on the "P68082" Entry

•     Browse through the page/s that follows. It will offer information on the structural/functional      features of the protein as well as references. You will see key headings on the left of the              webpage, find and click the “Sequence” heading. There you will see the amino acid sequence of myoglobin.

•     In the Sequence section, there will be a menu bar at the top. Click the “Tools” downward arrow and select “ProtParam”.

•    You will see that this tool allows you to investigate different parts of the protein.

•    We want to analyze the full-length polypeptide chain therefore click on … FT CHAIN 2-

154 Myoglobin.

•    The next page will show many different calculated/determined properties for the protein including the amino acid sequence, the molecular weight, pI, amino acid composition, etc.

(a) Record the molecular weight and theoretical pI value of myoglobin

(b) Under the amino acid composition table find and record the number of tyrosine and tryptophan residues found in myoglobin. Calculate the molar extinction coefficient for myoglobin at 280nm in   units of M(subunit)- 1cm-1 . Remember: tyrosine ε280nm  = 1490 M- 1  cm- 1  and tryptophan ε280nm  = 5500 M-   1  cm- 1

**Helpful Tip: Please refer to the introduction section for how to proceed with this calculation

Q5. (3 marks) In the same manner as you did for Myoglobin above, search “Catalase AND bovine” in the sameUniProtdatabase and then click on the "P00432" entry. Find the sequence section and then      query “ProtParam” in the dropdown menu box. Click on the full sequence “2 – 527” to analyze the    full amino acid composition of a monomer subunit.

(a) Record the molecular weight and theoretical pI value of catalase (monomer subunit)

(b) Under the amino acid composition table find and record the number of tyrosine and tryptophan residues found in a subunit of catalase and calculate the molar extinction coefficient at 280nm in     units of M(subunit)- 1cm-1 .

(c) Since we know that the native structure of catalase is a tetramer, use the molar extinction coefficient as determined in part (b) to calculate that of a catalase tetramer.

Q6. (6 marks) Now that you have calculated the molar extinction coefficients of myoglobin and catalase, you can use them to determine the protein concentration of your highest myoglobin-containing        fraction and your highest catalase containing fraction.

Step #1: Record the absorbance reading of each of the highest containing catalase and myoglobin fraction at 280 nm

Step #2: Determine the corrected absorbance value for the fraction of each protein above: To do this subtract the average baseline absorbance (make sure you indicate what this value is and how you arrived at it) from the above chosen values.

(Helpful Tip: Your baseline absorbance should be an average of a stable baseline seen prior to the elution of the first protein peak - you should be subtracting the same baseline value from both catalase and myoglobin).

Step #3: For the two corrected absorbance values calculated above, use the Beer-Lambert equation to determine the molar concentration of each catalase and myoglobin (using their respective            calculated ε280nm ). Assume that you used a cuvette with a 1.0 cm pathlength. (Show your                    calculations)

Step #4: Once you determine the concentration of each protein in the cuvette, factor in the dilution made (100 µL of the fraction added to 1000 µL of buffer) prior to reading the absorbance value of    the fraction. Determine the molar concentration of each protein in their respective fraction collected from the column chromatography.(show your calculations)

Step #5: Use the calculated MW from that recorded from the ProtParam database to convert the molar concentration for the catalase and myoglobin to their respective concentrations in units of "mg/mL". (Show your calculations)

Step #6: Consider the volume of the fraction collected for the highest containing catalase and myoglobin fraction and determine the "mg" amount recovered in them, respectively

Step #7: Fill in the table below with the results determined above:

Q7. (3 marks) Analyze Recovery:

(a) Compare the “mg” amount of catalase and myoglobin recovered (as calculated in Q#6) with the respective “mg” amount that was applied onto the column to start. Calculate the % recovery.

(b) In this experiment we expect a recovery of less than 50% because only one fraction of each          protein was retained. Explain what problem could arise if we decided to retain and pool three of the highest catalase-containing fractions and then three of the highest myoglobin-containing fractions - instead of only one of each, like we did in the lab.

Q8. (1 mark) Use your SDS-PAGE results to qualitatively analyze how well size exclusion chromatography separated the two proteins: Compare your results to what you see in the lane        containing the original protein mixture. Were the two proteins successfully separated? What would you see if the two proteins weren’t successfully separated via the size exclusion chromatography?

Q9. (2 marks)

(a) State the experimental molecular weights of catalase and myoglobin, determined from SDS- PAGE (Results: B-4)

(b) How do these values compare with the MW values obtained from the ProtParam database? Comment on the % error and show your calculations.

(c) Why is the MW of Catalase, as determined by SDS-PAGE, approximately ¼ of the native molecular weight as stated in the introduction?