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Format of Schedule Entries:
Topic / Technique

Work Due

Reading Assignment:
Please read and study all handouts and assignments before coming to lab.

Lab check-in; discussion of lab practices
Growing Protein Crystals / Vapor Diffusion from Sitting Drops

Work Due
• Prelab Exercise -- See lab handout. For help with this execise, read Horton, Sections 2.8, 2.9, 2.10.
NOTE: Prelab Exercises are individual work, and must to done BEFORE prelab. Accepted ONLY by 1:15 PM on lab days -- not accepted late.

Click HERE for lab handout.
Also read about pH and buffers in your class text.

For this first meeting, plan to prepare buffer at highest needed concentration, and discuss plans for first trials, which you will set up next week.

BEFORE next meeting, do all calculations of quantities for first trials. Come with a detailed plan for first trials.

Growing Protein Crystals / Vapor Diffusion from Sitting Drops
Continued

Preliminary crystallization report due 10/2.
Final Report (#1) due at lab on 11/6.

Set up first crystal trials this week. Let you instructor check your calculations of quantities to use in each well.

Use this Excel spreadsheet to check your calculations.

Protein Fingerprinting / Part I: Reducing, Digesting, and Lyophilizing Lysozyme
NOTE: Plan to come in Tuesday to take off dialysis and begin enzymatic digestion (less than one hour), and Wednesday to begin lyophilization (less than one hour). GR will stop lyophilizer and refrigerate samples Thursday morning.

Work Due
• Prelab Exercise -- See lab handout

Click HERE for lab handout.
Also read about enzymatic digestion of proteins in your class text.

Instrument Instructions

HPLC
Lyophilizer
UV/Vis Spectrophotometer

CRYSTALS: You will make first crystal-quality assessments this week, and then continue working on this project for six more weeks, interspersing it with your work on other projects.

Protein Fingerprinting / Part II: High Performance Liquid Chromatography (HPLC), Electronic (UV/Visible) Spectroscopy
NOTES:
1. ALL STUDENTS meet 1:15-2:00 PM for introduction to HPLC and to our instrument.
2. Each student team will then need a two-hour appointment to use the instruments between the dates of 10/2 and 10/6. Only one lucky student pair can do this part starting around 2:00 PM during the normal lab period. Priority of scheduling determined by random draw.

Work Due
• Preliminary Report (informal): Lysozyme Crystallization
1) What range(s) of conditions gave promising crystals?
2) What range(s) of conditions do you still need to explore?

Purifying and Characterizing Lysozyme / Part 1. Determining Protein Concentration

Work Due
• Prelab Exercise -- See lab handout
• Report #2: Protein Fingerprinting

No Lab

Purifying and Characterizing Lysozyme / Part 2. Ion-Exchange/Gradient-Elution Column Chromatography

Work Due
• Brief Report: Purifying Lysozyme, Part 1. Protein Concentration

Click HERE for lab handout.

Purifying and Characterizing Lysozyme / Part 3. Determining Specific Activity and Level of Purification

Work Due
Prelab Exercise for Part 3

Click HERE for lab handout.

Purifying and Characterizing Lysozyme / Part 3. Determining Specific Activity and Level of Purification
Continued: Extra day of data collection. If you need more time for data collection, the lab will be open all day Tuesday.
Work Due
Report #1: Growing Protein Crystals

Purifying and Characterizing Lysozyme / Part 4. Assessing Chromatography and Determining Molecular Weights of Products by Polyacrylamide-Gel ElectrophoresisNOTE: Plan to come in Tuesday to stain your gel and read your results (about one hour).

Work Due
• Prelab Exercise: See lab handout.
• Report #3: Purifying Lysozyme,
Parts 2 and 3. Chromatography and Assays
I will evaluate and return these reports during the lab -- be sure to pick yours up before you leave lab. Revision, if necessary, due Monday 11/20.

Click HERE for lab handout.

Also read about electrophoresis in your class text.

Exploring Lysozyme Catalysis and Inhibition / Enzyme Kinetics
Each team will receive a specific assignment for data collection

Work Due:
• Report #4, Purifying Lysozyme, Part 4, Electrophoresis. Submit only the Figure and Graph called for at the end of the handout. Revised Parts 2 and 3, if necessary. The "Report in Publication Form" listed there is due in two weeks.

Click HERE for lab handout.

Also read about enzyme kinetics in your class text.

Enzyme Kinetics, continued data collection
IMPORTANT: Process all kinetics data from last week and bring to class today (printed Excel graphs) for discussion before further data collection.

If you need to collect more kinetics data after today, the lab will be open all day tomorrow.

Work Due
None

SwissPdbViewer Tutorial, sections 6 - 11, plus VVP, Chapter 6.
Also see USM Lab Assignment

Exploring Lysozyme and Other Proteins / Advanced Computer Modeling and Analysis
Lab check-out: Please leave your drawer and the lab at least as tidy as you found it on the first day. Also remove and clean or discard all of your materials from the refrigerator and the incubator. Thanks.

Work Due
• Report in Pubication Form on "Purifying and Characterizing Lysozyme."
I will evaluate these reports and return them at class on Wednesday. Revision, if necessary, due next Monday, 12/10.

SwissPdbViewer Tutorial, sections 6 - 11, plus VVP, Chapter 6.
Also see USM Lab Assignment

Modeling, continued: Troubleshooting

Work Due
• Report #5: Lysozyme Catalysis and Inhibition
• Revised Report in Publication Form, if necessary

Work Due
• Report #6: Hand in modeling files by noon.

NOTE: As stated in The Fine Print, absolutely no reports or other work accepted after the last day of regular classes, Friday, 12/12, noon.