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

Advanced Experimental Biosciences SPRING 2010

Lectures are on MONDAYS from 3:00-4:00 p.m. in [location TBA] unless otherwise indicated; labs are scheduled from 1-6 p.m. on Tuesday (section 001) or Wednesday (section 002) in the Biology basement BCB teaching labs. Pre-lab QUIZZES will be given promptly at either 3 p.m. for lecture OR 1 p.m. for lab-- if you show up late, even if it's only 5 minutes, OR miss the lecture then you do not get to take the quiz (and I cannot offer make-up quizzes)!

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 ONLY resource (i.e., you should not use printed web pages or handouts from previous labs). 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 "Bios 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: 12 or 13 January

Lecture Topics (on Monday)		Experimental Procedures
Project Overview & Team Assignments		Buffer preparation
Laboratory Notebook		ADA stability study
Enzyme stability and buffer selection
ADA assay

Day 2 Protein Extraction and Precipitation: 19 or 20 January (NO Monday lecture because of MLK Day)

Lecture Topics		Experimental Procedures
Protein extraction		Pouring of SEC column
Salt precipitation		Cell lysis & tissue homogenization
Bradford assay		Bradford assay
Size exclusion chromatography		Ammonium sulfate precipitation
		Assays of stability samples

Day 3 Size Exclusion Chromatography: 26 or 27 January

Lecture Topics		Experimental Procedures
Detection of ADA Activity		Size exclusion chromatography
Dialysis		Qualitative ADA activity assays
Buffers for ion exchange		Selection/preparation of anion exchange buffer
Protein purification tables		Dialysis

Day 4 Anion Exchange Chromatography: 2 or 3 February

Lecture Topics (on Monday)		Experimental Procedures
Anion exchange chromatography set-up		Anion exchange chromatography
Protein determination		Qualitative and quantitative ADA activity assays
Plagiarism		Desalting Q fraction

Day 5 SDS-PAGE and Electrophoretic Transfer: 9 or 10 February

Lecture Topics (on Monday)		Experimental Procedures
Research Paper overview		SDS-PAGE
Writing the Materials and Methods		Silver stain of SDS-PAGE gel
Electrophoresis applications		Electrophoretic transfer

Day 6 Isoelectric Focusing and Western Analysis: 16 for 17 February

Lecture Topics (on Monday)		Experimental Procedures
Writing the Introduction		Western blot development
Isoelectric focusing		Isoelectric focusing
Coupled enzyme assays		Activity stain of IEF gel
Western development

Day 7 Enzyme Kinetics: 23 or 24 February

Lecture Topics (on Monday)		Experimental Procedures
Writing the Results, Discussion, and Abstract		Kinetic studies
Spectrophotometry
Kinetic review

*Results/Discussion drafts are due by 3 p.m. on 11 March (Biology 211: Dr. Beason's inbox)
**Notebooks and final paper are due by 1 p.m. on 25 March (Biology 211: BIOS 311 box)

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 10 June 1999
Updated 6 November 2009

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