Course Description:  The course is an advanced laboratory course that focuses on techniques for the preparation and quantitative analysis of proteins and other macromolecules, presenting students with a broad spectrum of techniques, approaches and concepts of contemporary biochemistry. You will learn DNA purification and analysis, protein expression and quantification, enzyme purification, enzymatic characterization, chromatography, electrophoresis, and spectroscopic analysis. While you become skilled in all of these techniques, you will be designing your own experimental procedures and continually analyzing and evaluating your results. You will do all of this while demonstrating safe laboratory practices and keeping a complete and organized lab notebook.

Course Research Project: In this course we will be conducting a research project to study the interactions between two proteins well known in carbohydrate metabolism: malate dehydrogenase (MDH) and citrate synthase (CS).  You will research, create a hypothesis and design your own experiments to test the interactions of these two interesting proteins.

SYLLABUS AND ASSIGNMENT SCHEDULE – the schedule is tentative and will change based on the pace of the class. 

Day

Block

Activity Description

Date

1

 

 

I

Course Intro, Lab Book, Solutions and Dilutions Review

Sep 2

2

 

I

 

Spectroscopy & Plate Reader

Sep 7

3

 

I

 

Protein Assay, Intro to Project/Hypothesis

Sep 9

4

 

II

 

Intro to MDH, CS Protein Interactions

Sep 14

5

 

II

 

Recombinant Protein Expression and Purification

Sept 16

6

 

 

 

III

 

Bioinformatic and Protein Modeling

GR HawkDock E Files (Z=both chains of CS; Y both chains of mMDH)

C _Recomb HawkDock E Files (CS = A & B; mMDH = C & D) AvsC, BvsC, BvsD

Hints: in PDB files identify by the following initial seq hMDH (NAKVAVC) hCS (NKKD)

MDH active site (PVIGGHAGKT) CS (LHG), (GHA), & (VDA)

Sep 21

7

 

III

 

Bioinformatic and Protein Modeling

Sep 23, 28, 30

& Oct 5

8

 

III

 

MDH-CS Interaction Hypothesis Planning/Presentation

 

 

Oct 5

9

 

IV

 

Recombinant MDH, CS Protein Purification

 

Oct 7

10

 

IV

 

Recombinant MDH, CS protein assay & SDS PAGE and Westernblot Day I

Hypothesis Mini - Presentations (P1)

 

Oct 12

11

 

IV

 

Recombinant MDH, CS protein assay & SDS PAGE and Westernblot Day II

 

 

Oct 14

12

 

 

V

Experimental Design and Assay Development

  • Develope WesternBlot
  • Presentations (don't forget the peer evaluation)

Enzyme assay introduction and vernier/plate reader introdution

 

 

Oct 19

13

 

VI

 

Km and Vmax determination of MDH using plate reader assay

 

Oct 21

14

 

VI

 

Km and Vmax determination of MDH using plate reader assay

 

Oct 26

15

 

VI

 

Determination of MDH Specific Activity using 1.0 ml Assay

 

Oct 28

 

16

 

VI

 

Catch Up on Lab Book and Assignment Day

 

Nov 2

 

17

 

VI

 

MDH Kinetics

 

Nov 4

18

VII

MDH Kinetics

Nov 9

19

VII

Purification

Nov 11

20

VII

Kinetics of mutants

Nov 16

21

VII

Data Processing / Abstract

- GraphPad Instructions / GraphPad_Prism Support

- How to write an abstract

Nov 18

22

VII

Finalize Experimental Plans - TEV Cleavage and Pull Down Protocol and Expt Design

Nov 23

H

 

Thanksgiving – No class

Nov 25

23

VII

Independent project – test hypothesis

Nov 30

24

VII

Independent project – test hypothesis

Dec 2

26

VII

Independent project – test hypothesis

Dec 7

26

VII

Independent project – test hypothesis and Clean Up

Dec 9

 

 

Final/Presentation –

  • Final Presentation Instructions

Dec 14&16

Assignments Schedule:

 

                      Due Date

 

Pre-lab Assignments

 

 

Points

 

Pre-lab 1 (PL1) –Purification (bring to class)

 

Oct 7

12.5

 

Pre-lab 2 (PL2) –MDH Assay

 

Oct 21

12.5

 

 

 

Subtotal

25

Quizzes

 

 

 

 

Pop quizzes

 

N/A

50

 

Subtotal

50

Homework

 

 

 

 

HW1 –Biochem Lab Math and MDH

 

Sept 7

20

 

HW2 –Spectoscopy and Protein Assay

 

Sept 14

20

 

HW3 –Protein Expression and Purification

UPDATED Deadline

Sept 28

20

 

HW4 –Bioinformatics (same as posted day 7)

 

Oct 14

40

 

HW5 –MDH kinetic assays

 

Dec 1

10

 

 

Sub-total

125

Laboratory Notebook

 

 

 

 

Daily Notebook Checks (5 pts ea)

 

N/A

        100

 

Notebook Check I (NB1)

 

Oct 12

20

 

Notebook Check II (NB2)

 

Nov 16

30

 

Notebook Check III (NB3)

 

Dec 14

50

 

 

Sub-total

200

Project-related Assignments

 

 

 

 

P1 – Basic Hypothesis – presentation, group

 

Oct 19

20

 

P2 – Experimental design – written and presentation Group

 

 

 

 

P3 – Short presentation – In class, group

 

  

 

 

P4 – Progress report – Group (+ instr/peer evals)

 

 

 

 

P5 – Final presentation

 

Dec 14

100

 

 

 

 

Writing Assignments

 

 

 

 

W1 – Introduction & hypothesis and re-write

 

Oct 21

40 (30/10)

 

W2 - Abstract

 

Nov 22

10

 

W3 –Figures and Legends

 

Dec 7

20

 

 

 

 

 

 

W4 – Results/Discussion

 

Dec 11

40

 

 

 

 

 

 

 

 

 

Total Points

 

 

 

Optional/Background Links and Tutorials on Protein-Protein Interactions

STRING Prediction Tutorial