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Labs: M 1:30-5pm / F 12:30-4pm / 300 SCI
Mona Hall Office: 287 /
x3057 / office hours will be announced
The lab is a project-based one, focused on the enzyme lysozyme. You will carry out two extended projects over the course of the semester: purification of the enzyme and use of advanced instrumentation to characterize the enzyme. Each project culminates in a written and/or oral presentation of your results.
Lab Schedule
|
Week
|
Monday |
Thursday
|
Friday
|
Laboratory
|
|
1
|
Sept 8 |
Sept 11
|
Sept. 12
|
Introduction and enzyme assay |
|
2
|
Sept 15 |
Sept 18
|
Sept 19
|
Discussion of purification methods. Decision on methods and variations to be used by each team. Make buffers and equilibrate resins. |
|
3
|
Sept 22 |
Sept 25
|
Sept 26
|
Purification begins: Filter egg-white and perform ion-exchange chromatography. |
|
4
|
Sept 29 |
Oct 2
|
Oct 3
|
Purification continues: Collect fractions. |
|
5
|
Oct 6 |
Oct 9
|
Oct 10
|
Protein and enzyme assays of fractions. |
|
6
|
Oct 20 |
Oct 16
|
Oct 17
|
SDS-PAGE and Discussion of purification results |
|
7
|
Oct 27 |
Oct 23
|
Oct 24
|
Project on protein methods begins with Rotation I Notebooks due. |
|
8
|
Nov 3 |
Oct 30
|
Oct 31
|
Protein methods continues with Rotation II |
|
9
|
Nov 10 |
Nov 6
|
Nov 7
|
Independent project: Variation on a protein method |
|
10
|
Nov 17 |
Nov 13
|
Nov 14
|
Independent project continues. |
|
11
|
Nov 24 |
Nov 20
|
Nov 21
|
Write short paper |
|
12
|
Dec 8 |
Dec 4
|
Dec 5
|
Presentation of project results Final paper due 3 days after last lab |
Pre-lab assignments
In order for everyone to be prepared for
the lab, the pertinent section for each week's lab should be read before
lab. In addition, the assignments described below should be completed.
Written work should be included on the left-hand side of your notebook
and xeroxed prior to class. The xeroxed copy will be collected and evaluated
by the instructor.
|
Week
|
Assignment
|
|
1
|
Bring lab manual and notebook to lab. |
|
2
|
Calculate the specific activities of the lysozyme standard. Read the textbook section on lysozyme. |
|
3
|
Outline the approach you will take to purify lysozyme, using a flow diagram. For a batch method, indicate if lysozyme will reside in the supernatant or pellet. For a column separation, indicate the likely contents of the flow-through and subsequent eluted fractions. |
|
4
|
Review the progress made during the first week of purification and decide on your approach for this week (e.g. method of column elution) |
|
5
|
Prepare a dilution scheme for BSA, the 2 mg, mL standard for the protein assay. Decide on the dilutions of your unknown for the protein assay. Attempt three different dilutions to improve your chances that the results will fall on the standard curve. For the enzyme assay, decide on dilutions of your unknowns that contain protein. (If you expect no lysozyme in a fraction, run an undiluted sample.) |
|
6
|
Graph the standard curve for the protein assay and complete the calculations for all assays and a data summary table of your results. Be ready to report to the class the specific activity, total activity, % yield, and fold purification of your fractions. |
|
7
|
Read the sections in the manual on your assigned protein method and also read the listed references. NOTEBOOKS ARE DUE. |
|
8
|
Read the section in the manual on your assigned protein method and also read the listed references. Schedule a meeting with your instructor three days after lab to discuss independent project and bring a materials list. |
|
9
|
Complete a pre-lab plan in your notebook for your independent project (e.g., calculate how to prepare appropriate volumes of buffers, reagents, enzymes, etc.) |
|
10
|
Complete a pre-lab plan in your notebook for this second week of lab work |
|
11
|
Schedule a 20-min meeting with your instructor to discuss your results. Use the remaining lab time to write your scientific paper. |
|
12
|
Presentation of your independent project. Bring a copy of your draft to class for peer review. |
Reading Period: FINAL PAPER DUE. Also turn in the peer review forms completed by other students and your first draft.