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

BIOC 311 Advanced Experimental Biosciences SUMMER 2016

Lab meets from 9 AM - 4 PM on M-F for 6 sessions; 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. After the last lab meeting, we will meet from 1-2 pm on T,W, and R so you have opportunities to ask questions about data analysis/interpretation and the sections of the paper.

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 Protein Extraction and Precipitation: Mon., 23 May


Experimental Procedures

Honor Code Policy

SEC column preparation
Team library project

Cell lysis & tissue homogenization

Enzyme stability and assays

Bradford assay

Protein extraction

ADA assay

Bradford assay

Ammonium sulfate precipitation

Salt precipitation


Day 2 Size Exclusion Chromatography: Tues., 24 May


Experimental Procedures

Research Paper overview

Size exclusion chromatography


Qualitative ADA activity assays

Size exclusion chromatography theory

Measurement at 280 nm

Detection of ADA Activity

Selection/preparation of anion exchange buffer

Buffers for ion exchange




Day 3 Isoelectric Focusing: Wed., 25 May


Experimental Procedures

Writing the Materials and Methods

Isoelectric focusing (IEF)

Isoelectric focusing

ADA activity stain of IEF gel

Coupled enzyme assays

Silver stain of IEF gel

Day 4 Anion Exchange Chromatography: Thurs., 26 May


Experimental Procedures

Writing the Introduction

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 SDS-PAGE, Electrophoretic Transfer, and Western Blot Development: Fri., 27 May


Experimental Procedures

Writing the Results


Electrophoresis applications

Silver stain of SDS-PAGE gel

Western blot development

Electrophoretic transfer

Western blot development

Day 6 Enzyme Kinetics: Tues., 31 May


Experimental Procedures

Writing the Discussion and Abstract

Kinetic studies


Kinetic review


Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 25 February 2010
Updated 6 April 2016