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

BIOC 311 Advanced Experimental Biosciences SPRING 2014

Discussions, which are required, are on MONDAYS from 3:00-4:00 p.m. in Duncan College (DCC) 113 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 labsPlease bring at least 1-2 computers per team to each discussion and lab.

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. Please check OWL-Space for the Release/Due Dates; 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

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: 14 or 15 January


Experimental Procedures

BIOC 311 Honor Code Policy

Buffer preparation

Project Overview & Team Assignments

ADA stability study

Enzyme stability and buffer selection

ADA assay

Day 2 Protein Extraction and Precipitation: 21 or 22 January (NO Monday meeting because of MLK Day)


Experimental Procedures

Protein extraction

Pouring of SEC column

Salt precipitation

Cell lysis & tissue homogenization

Bradford assay

Bradford assay

Pre-lab: ADA Elevator Talk

Ammonium sulfate precipitation

Assays of stability samples

Day 3 Size Exclusion Chromatography: 28 or 29 January


Experimental Procedures

Size exclusion chromatography theory

Size exclusion chromatography

Detection of ADA activity

Qualitative ADA activity assays


Selection/preparation of anion exchange buffer

Buffers for ion exchange


ADA stability study results

Day 4 Anion Exchange Chromatography:4 or 5 February


Experimental Procedures

Plagiarism and Research Paper overview

Anion exchange chromatography

Writing the Materials and Methods

Qualitative and quantitative ADA activity assays

Anion exchange chromatography set-up

Desalting Q fraction

Protein determination

Protein purification tables

Day 5 Enzyme Kinetics: 11 or 12 February


Experimental Procedures

Writing the Introduction

Kinetic studies


Kinetics review

Day 6 SDS-PAGE and Electrophoretic Transfer: 18 or 19 February


Experimental Procedures

Writing the Results


Electrophoresis applications

Silver stain of SDS-PAGE gel


Electrophoretic transfer

Day 7 Isoelectric Focusing and Western Blot Development: 25 or 26 February


Experimental Procedures

Writing the Discussion and Abstract

Western blot development

Isoelectric focusing

Isoelectric focusing

Coupled enzyme assays

Activity stain of IEF gel

Western blot development


Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 10 June 1999
Updated 3 January 2014