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 labs. Please 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.

Preparation		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.

Preparation		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

Preparation		Experimental Procedures
Size exclusion chromatography (SEC)		Size exclusion chromatography
Detection of ADA activity		Qualitative ADA assays
Dialysis		Preparation of anion exchange buffer
Buffers for ion exchange		Dialysis
ADA Presentations
ADA stability results

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

Preparation		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.

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: 27, 28, or 29 Sept.

Preparation		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.

Preparation		Experimental Procedures
Writing the Discussion and Abstract		Enzyme kinetics studies
Spectrophotometry
Enzyme kinetics review

POST-LAB DUE DATES

Final notebook pages (including data analysis/conclusions and Table of Contents) are due by 4 pm on Tues., 18-Oct-2016 (ABL 326: Dr. Beason's inbox or BIOC 311 box); please use a clip to keep your pages together
Results draft is due by 4 pm on Tues., 1-Nov-2016 (ABL 326: Dr. Beason's inbox). *Please use a paper clip; do not use staple pages.
Discussion draft is due by 4 pm on Tues., 8-Nov-2016 (ABL 326: Dr. Beason's inbox). *Please use a paper clip; do not staple pages.
Final paper (including graded drafts) is due by 4 pm on Tues., 29-Nov-2016 (ABL 326: Dr. Beason's inbox or BIOC 311 box). *Please use a folder with pockets (print your name and lab day on the front of the folder); do not use staples or paper clips on the final paper.

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

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