Purification and Comparison of Mouse Adenosine Deaminase
from Recombinant and Native Sources

BIOC 311 Advanced Experimental Biosciences SUMMER 2013

Class is from 9 AM - 4 PM on M,T,W,F; there is a 45-60 min. break for lunch. We meet in the Biology basement BCB teaching labs. Please bring at least 1-2 computers per team to each lab meeting.

Pre-lab exercises are submitted in OWL-Space using the "Tests & Quizzes" tool: although there is no time limit for completing the exercises, you are allowed a single submission. The pre-lab for each lab will close at 8:30 am on the day of lab (check OWL-Space for "open" dates/times); you must submit your work during this time frame to get full credit for the pre-lab. You must complete the exercises ALONE (i.e., do not work on the pre-lab with anyone else): you may use your class notes/web site/Scopes; have a calculator and pen/pencil with scratch paper on hand as you answer the questions.

You must come to lab prepared--this requires you to READ the experimental protocols on the course web site BEFORE coming to lab, not just print a copy of them and bring it with you. Bring only the information you need to perform the experiments. The procedures for each day are available here and you will be given any additional information in the pre-lab lectures.

SPECIAL NOTE: Record enough procedure details in your notebook during each day of lab so that you can repeat these procedures using your notebook as the MAIN resource. An exception to this note are the instructions for using the spectrophotometers; there are 3 models, and since you may not always use the same type, you can have those instructions handy in your "BIOC 311 Binder" (for loose data).

In this laboratory course, we use several pieces of equipment and many different reagents from Bio-Rad. Check out their web page for useful information on life science research products. On certain lab days you may want to read specific Bio-Rad manuals.

Day 1 Biological Buffers: Tues., 14 May

Preparation		Experimental Procedures
Honor Code Policy and Plagiarism		Selection/preparation of anion exchange buffer
Team library project		ADA assays
Enzyme stability and buffer selection
Buffers for ion exchange
ADA assay

Day 2 Protein Extraction and Precipitation: Wed., 15 May

Preparation		Experimental Procedures
Research Paper overview		SEC column preparation
Protein extraction		Cell lysis & tissue homogenization
Bradford assay		Bradford assay
Salt precipitation		Ammonium sulfate precipitation

Day 3 Size Exclusion Chromatography: Fri., 17 May

Preparation		Experimental Procedures
Size exclusion chromatography theory		Size exclusion chromatography
Detection of ADA Activity		Qualitative ADA activity assays
Dialysis		Measurement at 280 nm
Elevator talks		Dialysis
Writing the Introduction

Day 4 Anion Exchange Chromatography: Mon., 20 May

Preparation		Experimental Procedures
Writing the Materials and Methods		Anion exchange chromatography
Anion exchange chromatography set-up		Qualitative and quantitative ADA activity assays
Protein determination		Measurement at 280 nm
Protein purification tables		Desalting Q fraction

Day 5 Enzyme Kinetics: Tues., 21 May

Preparation		Experimental Procedures
Writing the Results		Kinetic studies
Spectrophotometry
Kinetic review

Day 6 SDS-PAGE, Electrophoretic Transfer, and Western Blot Development: Wed., 22 May

Preparation		Experimental Procedures
Writing the Discussion		SDS-PAGE
Electrophoresis applications		Silver stain of SDS-PAGE gel
Western blot development		Electrophoretic transfer
		Western blot development

Day 7 Isoelectric Focusing: Fri., 24 May

Preparation		Experimental Procedures
Writing the Abstract		Isoelectric focusing (IEF)
Isoelectric focusing		ADA activity stain of IEF gel
Coupled enzyme assays		Silver stain of IEF gel

DUE DATES for NOTEBOOK PAGES and RESEARCH PAPER

Notebook Pages
- In-lab notebook pages are due at the end of each lab day (put in your BIOC 311 folder in Biology basement)
- Final notebook pages (including data analysis/conclusions and Table of Contents) are due by 4 pm on Mon., 27 May (ABL 326: Dr. Beason's inbox); please use a clip to keep your pages together
Research Paper
- Introduction draft is due at 9 am on Mon., 20 May (turn in at the beginning of lab). *Please use a paper clip; do not staple pages.
- Materials and Methods draft (days 1-5) is due at 9 am on Wed., 22 May (turn in at the beginning of lab). *Please use a paper clip; do not staple pages.
- Results and Discussion drafts are due by 4 pm on Wed., 29 May (ABL 326: Dr. Beason's inbox). *Please use a paper clip; do not staple pages.
- Final paper is due by 9 am on Wed., 5 June (ABL 326: Dr. Beason's inbox). *Please use a folder with pockets; do not use staples or clips.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 25 February 2010
Updated 11 April 2013

Purification and Comparison of Mouse Adenosine Deaminase from Recombinant and Native Sources