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

Advanced Experimental Biosciences FALL 2008

Lectures are on MONDAYS from 3:00-4:15 p.m. in Geology Lab (GL) 100 unless otherwise indicated; labs are scheduled from 1-7 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. or 1 p.m.-- if you show up late then you do not get to take the quiz.

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: 26 or 27August

Lecture Topics		Experimental Procedures
Project Overview		Buffer preparation
Laboratory Notebook		Accuracy and precision check of pipettors
Enzyme stability and buffer selection		ADA stability study
Pre-lab lecture

Day 2 Protein Extraction and Precipitation: 2 or 3 September (Lecture given at beginning of lab)

Lecture Topics		Experimental Procedures
Protein extraction and precipitation		Cell lysis
Bradford assay		Bradford assay
Chromatography introduction		Ammonium sulfate precipitation
Pouring size exclusion chromatography (SEC) column		Assays of stability samples
		Pouring of SEC column

Plagiarism Workshop: Mon., 8 September, 3 p.m.

Day 3 Size Exclusion Chromatography: 9 or 10 September (Lecture given during lab)

Lecture Topics		Experimental Procedures
ADA Stability Study results		Size exclusion chromatography
Buffers for ion exchange		Qualitative ADA activity assays
Detection of ADA Activity		Selection/preparation of anion exchange buffer
Dialysis		Dialysis
Purification Tables

Day 4 Anion Exchange Chromatography: 16 or 17 September

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

Day 5 SDS-PAGE and Electrophoretic Transfer: 23 or 24 September

Lecture Topics		Experimental Procedures
Research Paper overview		SDS-PAGE
Writing the *Materials & Methods*		Coomassie stain
Electrophoresis applications		Electrophoretic transfer

Day 6 Isoelectric Focusing and Western Analysis: 30 September or 1 October

Lecture Topics		Experimental Procedures
Writing the *Introduction*		Western blot development
Isoelectric focusing		Isoelectric focusing
Coupled enzyme assays		Activity stain
Silver stain		Silver stain
Western development

Day 7 Enzyme Kinetics: 7 or 8 October

Lecture Topics		Experimental Procedures
Writing the *Results, Discussion, and Abstract*		Kinetic studies
Spectrophotometry
Kinetic review

*Results/Discussion drafts are due by 1 p.m. on 15 or 16 October
**Notebooks and final paper are due by 1 p.m. on 29 or 30 October

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 10 June 1999
Updated 28 August 2008