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

BIOC 311 Advanced Experimental Biosciences FALL 2014

Discussions, which are required, are on MONDAYS from 3:00-4:30 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 labs. Please 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, 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: 26 or 27 August

Preparation		Experimental Procedures
BIOC 311 Honor Code Policy		Buffer preparation
Project Overview & team library project		ADA stability study
Enzyme stability and buffer selection
ADA assay

Day 2 Protein Extraction and Precipitation: 2 or 3 September (NO Monday discussion because of Labor Day)

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

Day 3 Size Exclusion Chromatography: 9 or 10 September

Preparation		Experimental Procedures
Detection of ADA activity		Size exclusion chromatography
Dialysis		Qualitative ADA activity assays
ADA stability results		Selection/preparation of anion exchange buffer
Buffers for ion exchange		Dialysis

Day 4 Anion Exchange Chromatography: 16 or 17 September

Preparation		Experimental Procedures
Research Paper overview and Plagiarism		Anion exchange chromatography
Writing the Materials and Methods		Qualitative and quantitative ADA activity assays
Anion exchange chromatography set-up		Desalting Q fraction
Protein purification tables

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

Preparation		Experimental Procedures
Writing the Introduction		SDS-PAGE
Electrophoresis applications		Silver stain of SDS-PAGE gel
		Electrophoretic transfer

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

Preparation		Experimental Procedures
Writing the Results		Western blot development
Isoelectric focusing		Isoelectric focusing
Coupled enzyme assays		Activity stain of IEF gel
Western blot development

Day 7 Enzyme Kinetics: 7 or 8 October

Preparation		Experimental Procedures
Writing the Discussion and Abstract		Kinetic studies
Spectrophotometry
Kinetic review

POST-LAB DUE DATES

Final notebook pages (including data analysis/conclusions and Table of Contents) are due by 3 pm on Thurs., 16 October (ABL 326: Dr. Beason's inbox or BIOC 311 box); please use a clip to keep your pages together
Results draft is due by 3 pm on Thurs., 24 October (ABL 326: Dr. Beason's inbox).
Discussion draft is due by 3 pm on Thurs., 31 October (ABL 326: Dr. Beason's inbox).
Final paper is due by 3 pm on Mon., 18 November (ABL 326: Dr. Beason's inbox or BIOC 311 box). *Please use a folder with pockets; do not use staples or clips.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 10 June 1999
Updated 2 May 2014

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