Lecture and Lab Experiment Schedule (Fall 2009)

 

 Pease Note: For each lab session:

  1. Please thoroughly read the appropriate material in Fundamental Approaches for Biochemistry and Biotechnology by Ninfa,Ballou, and Benore (NB&B).
  2. Check the Lecture and Laboratory Notes webpage for additional information.
  3. Prepare a Flow Chart for each day's experiments (see Course Information: Flow Chart).
  4. You will need Adobe Acrobat in order to download protocols that are not covered by your text.

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Important Administration Dates

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Sep. 18

Last day to drop with a Deletion

Oct. 16

Last day to drop with a "W"

Date

Experiment

Textbook Reading

Protocol

Special Notes

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Tu,Aug.25

Introduction to BIOC 463a: Orientation, Course Objectives, and Expectations.

 

 Sections 1.1 - 1.7 in NB&B contain some very useful information.

Please read Syllabus thoroughly.

 

Lecture will meet in Shantz 242E for morning class and BSW 237 (to be changed) for afternoon class. We will journey over to Koffler 540 to acquaint ourselves with the lab after Lecture.

Th,Aug.27

Preparation and Characterization of Buffers.

Sections 2.6 and 2.7 in NB&B.

Exp_1.pdf

Lecture Notes for Expt. 1.

Please have Tables filled out prior to coming to class for all but the raw data. Make sure you have done the calculations for ALL the Tables in Expt. 1-1 and 1-2 PRIOR to coming to class! We will check to make sure this has been done before you can begin working on the exercise.

Lab Reports: For the first Lab Report follow the instructions at the end of the protocol for the construction of the figures and their discussionFor a more general set of instructions see Lab Report.pdf.

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Tu,Sep.1

Absorbance Spectra of PNPOH and PNPO-.

Detemination of the Extinction Coefficient for PNP.

Ch.3: Sections 3.1 - 3.5. Section 3.7 should be review of O. Chem, while Sec. 3.8 will be covered in detail at a later date.

Exp_2.pdf

Lecture Notes for Expt. 2.

The phosphate and Tris buffers prepared in Expt. 1 will be used in this experiment.

 

Th,Sep.3

Spectrophotometric determination of the pKa of PNP in Phosphate Buffer and the Effect of Alcohol on the pKa.

Ch.3

Exp_2.pdf

For the second Lab Report you will follow the instructions handed out in class for submitting a manuscript to the journal Biochemistry.

Tu,Sep.8

Colorimetric Determination of Protein Concentation: Lowry, BCA, and Bradford Assays.

Ch.4 (Sections 4.1 - 4.4)

Exp_3.pdf

Lecture Notes for Expt. 3.

Th,Sep.10

Determination of Protein Concentration: A280 Method and the Effect of DNA on the A260/A280 ratio.

Determination of Protein Concentration: Heme Chromophore Methods

Ch. 4

Exp_3.pdf

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Tu,Sep.15

Size Exclusion and Ion Exchange Chromatography

Ch.5: Sections 5.1, 5.2, and 5.4

Exp_4.pdf

Lecture Notes for Expt. 4

This will be a long and challenging lab, come prepared!

Th,Sep.17

Affinity Chromatography (Beta-galactosidase purification)

Ch.5: Sec. 5.3

Exp_4.pdf

This will also be a very challenging experiment. It is important to note that ALL of the buffer you will be working with contain BME! Therefore, review the Colorimetric material to decide which colorimetric assay you should use to quantitate the enzyme concentration.

Tu,Sep.22

SDS-PAGE

Ch.6 (All sections)

Expt_5.pdf

.Lecture Notes for PAGE

Th,Sep.24

Native PAGE and Performing Enzyme Assays on a native gel.

Ch.6. Native gels are discussed in Section 6.5

Expt_5.pdf

We will assay for the presence of beta-galactosidase and alkaline phosphatase on the same gel. For beta-gal, we will use the same ONPG assay as before.

Tu,Sep.29

 

Western Transfer of Alkaline Phosphatase

Day 1: Running the SDS-PAGE gel and doing the Western Transfer.

We will hand out the reading material for this procedure in class.

Western Blots.pdf

We will begin class in Koffler 540 at 8 am and 1 pm.

There are no official lecture notes, however a lot of background information will be in the handout.

Th,Oct.1

Western Transfer of Alkaline Phosphatase

Day 2: Binding of Antibody and performing the colorimetric assay.

 

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Tu,Oct.6

Th,Oct.8

 

Mass Spectrometry and Proteomics

Sec. 3.8: Mass Spectrometry

Sec. 6.5: Isoelectric focusing, 2D gel electrophoresis

pps. 98 - 100 in Lehninger Principles of Biochemistry

All in class information will be online, see links on homepage.

Class will begin at 9 am and 1 pm in BSW 243.

 

Tu,Oct.13

 

Ligand Binding Equilibrium (Avidin and HABA)

 

Ch.11 in NB&B: This is one of the BEST treatises on ligand binding that you will every encounter!

Expt. 10.pdf

Lecture Notes for Ligand Binding

Helpful Hints: (1). Most Kd values fall into the range of 10(^-6) and 10(^-5) M range. (2). Keep A500 for HABA bound to avidin <= 0.2. (3) The volume of buffer + protein before adding HABA will be 1 mL. (4) [Avidin] in terms of the tetramer is 36.8 uM and each subunit contains a single ligand binding site. Therefore calculate the Receptor concentration. (5) You want the [Receptor] to be <= Kd.

We will first do a titration of Avidin using HABA, from which you can determine Kd.

Note: non-specific binding ( Ch. 11 in NB&B) may be observed. Your [LR] needs to be corrected if seen.

Kd will be determined from Double Reciprocal plot, Scatchard plot, and Non-linear regression analysis of hyperbolic data using ORIGIN.

 

Th,Oct.15

Competitive Ligand Binding Assay:

Back Titration of HABA:Avidin complex with Desthiobiotin (DTB).

Competitive ligand section of NB&B

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Once Kd for HABA has been determined, you will set up a competitive ligand experiment where [HABA]> >> Kd. Why is this important? The HABA:Avidin complex will then be titrated with Desthiobiotin (DTB), from which it should be possible to determine a value for Kd for DTB.

Tu,Oct.20

Th,Oct.22

Review for Exam (please come with specific questions).

Two-Thirds Exam (~60 minutes)

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For Review and Exam we will meet:

Morning class: 9 am in Koffler 540

Afternoon class: 1 pm in Koffler 540

Tu,Oct.27

 

Th,Oct.29

Alkaline Phosphatase (AP) Purification: Stage 1 Enzyme, Spheroplasts and Activity Assays.

AP Purification: Stages 2 and 3 Enzyme by Heat and AmSO4 Precipitation.

Ch. 7: Gen. Protein Purrification

Ch. 8: Sections 8.1 and 8.2 for prokaryotic cells.

Ch. 9: AP purification.

Expt. 7-1 (N&B). Day One: Stage 1 Enzyme

Day Two: Stage 2 and 3 Enzymes.

Lecture Notes: Protein_purification.pdf

Make a copy of the Table on p. 207 prior to class.

Tu,Nov.3

AP Purification: Stage 4 Enzyme by DEAE Chromatography.

 Ch.9

 Day Three: Stage 4 Enzyme.

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Th,Nov.5

AP Concentration Determination, Activity Assays, SDS-PAGE, and Mass Spectrometry.

Ch.9

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Review the material on Bradford Assays and pouring SDS-PAGE (10%) gels

Tu,Nov.10

Steady-State Kinetics: Determination of Km, Vmax, and kcat of AP

Ch. 10: Sec. 10.1-10.4

Expt. 8-1

Lecture Notes: Steady_State_Kinetics.pdf

Th,Nov.12

Steady-State Kinetics: Competitive Inhibition by Inorganic Phosphate

 

Over the weekend: EDTA inhibition and using kinetic data to propose a mechanism

Ch. 10: Sec. 10.5.

Download ap_edta.pdf file from homepage and work on it over the weekend.

Expt. 8-2. The protocol given in N&B is for a Dixon method of studying competitive inhibitors. Consult your BIOC 462 text for a Lineweaver Burke method.

 

Half of the class will do a Lineweaver-Burke (seen in most textbooks) type of competitive Inhibitor experiment (measure v0 as fcn of [S], holdingI [I] constant. Other half will do procedure given in N&B, which is a Dixon method, where v0 is determined as fcn of varying [I], while holding [S] constant. Both methods give Ki for inorganic phosphate. We will compare Ki values obtained by both methods in next class.

The ap-edta.pdf exercise is to be worked on at home over the weekend. Follow the instructions for data analysis and come to class prepared to suggest a set of experiments you would use to determine the mechanism of edta inactivation of AP.

Tu,Nov.17

Special AP research Project.

Too many to list

You will design and carry out your own experiment as discussed in class.

This project originates with the native gel experiment performed earlier in the semester where the effect of BME on AP activity was observed. Each group will get to choose their approach to answering the question: What is protein denaturation?

In addition, we will have projects based on kinetic activity of the enzyme.

Th,Nov.19

Special AP Research Project

See above

See above

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Tu,Nov.24

Presentation of AP Research Project Results

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Each group will give a short (<= 10 minute) presentation of the results from their project.

Tu,Dec.1

Th,Dec.3

Research Presentations

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Tu,Dec.1 - Tu,Dec.8

Research Presentations

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Th,Dec 10

Due Date for Final AP Manuscript

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