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

BIOC 311 Advanced Experimental Biosciences FALL 2016

Discussions, which are required, are on MONDAYS from 3:00-4:00 p.m. in Herring (HRG) 129 unless otherwise indicated; labs are scheduled from 1-6 p.m. on Tuesday (section 001) , Wednesday (section 002), or Thursday (section 003) 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 Canvas using the "Quizzes" tool: although there is no time limit for completing the exercises, you are allowed a single submission. Please check Canvas for the Release/Due Dates; you must submit your work during this time frame to get 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, etc.; 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: 23, 24, or 25 Aug.


Experimental Procedures

Honor Code Policy

Buffer preparation

Project Overview & team library project

ADA stability study assays

Enzyme stability and buffer selection

ADA assay

Day 2 Protein Extraction and Precipitation: 30 or 31 Aug. or 1 Sept.


Experimental Procedures

Protein extraction

Pouring of SEC column

Bradford assay

Crude extract isolation

Salt precipitation

Bradford assay

SEC column preparation

Ammonium sulfate precipitation

ADA stability study assays

Day 3 Size Exclusion Chromatography: 6, 7, or 8 Sept.

NO Monday meeting because of Labor Day


Experimental Procedures

Size exclusion chromatography (SEC)

Size exclusion chromatography

Detection of ADA activity

Qualitative ADA assays


Preparation of anion exchange buffer

Buffers for ion exchange


ADA Presentations

ADA stability results

Day 4 Anion Exchange Chromatography: 13, 14, or 15 Sept.


Experimental Procedures

Research Paper overview and Plagiarism

Anion exchange chromatography

Writing the Materials and Methods

Qualitative/quantitative ADA assays

Anion exchange chromatography set-up

Desalting Q fraction

Protein Purification Tables

Day 5 SDS-PAGE and Electrophoretic Transfer: 20, 21, or 22 Sept.


Experimental Procedures

Writing the Introduction


Electrophoresis applications

Silver stain of SDS-PAGE gel


Electrophoretic transfer

Day 6 Isoelectric Focusing and Western Blot Development: 27, 28, or 29 Sept.


Experimental Procedures

Writing the Results

Western blot development

Isoelectric focusing

Isoelectric focusing (IEF)

Coupled enzyme assays

Activity stain of IEF gel

Western blot development

Day 7 Enzyme Kinetics: 4, 5, or 6 Oct.


Experimental Procedures

Writing the Discussion and Abstract

Enzyme kinetics studies


Enzyme kinetics review


Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 10 June 1999
Updated 22 August 2016