Lecture and Lab Experiment Schedule (Spring 2012)

 

 Pease Note: For each lab session:

  1. Please thoroughly read the appropriate material in Fundamental Approaches for Biochemistry and Biotechnology, 2nd Ed. 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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Last day to drop with a Deletion

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Last day to drop with a "W"

Date

Experiment

Textbook Reading

Protocol

Special Notes

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Th,Jan 12

Introduction to BIOC 463a

Preparation and Characterization of Buffers.

Lecture Notes for Expt. 1

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

Sections 2.6 and 2.7 in NB&B.

Exp_1.pdf

Make sure you have thoroughly read the course Syllabus

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.

Tu,Jan 17

Absorbance Spectra of PNPOH and PNPO-.

Detemination of the Extinction Coefficient for PNP.

Lecture Notes for Expt. 2.

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

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

Th,Jan 19

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,Jan 24

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

Lecture Notes for Expt. 3.

Ch.4 (Sections 4.1 - 4.4)

Exp_3.pdf

Th,Jan 26

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,Jan 30

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,Feb 2

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,Feb 7

SDS-PAGE

Ch.6 (All sections)

Expt_5.pdf

Lecture Notes for PAGE

Th,Feb 9

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,Feb 14

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,Feb 16

Western Transfer of Alkaline Phosphatase

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

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Class will meet in Koffler 540 at 8 am and 1 pm.

Tu,Feb 21

Th,Feb 23

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,Mar.1

Th,Mar.3

Ligand Binding: Determination of Kd for binding of HABA by avidin. Ch. 10 in N&B, Ch. 11 in NB&B. This is one of the best treatments of the math and the methods used for doing ligand binding assays that you will find!

There is no protocol, you will have to design your own!

See notes ==>

Precision is the key to success in this experiment!

Lecture notes: ligand_binding.pdf

In this experiment you will have to make an intelligent guess (1< [L] < 10 uM) about the value for Kd (the paradox) since you will have to titrate the receptor with the ligand, getting some data points less than Kd, and going to sufficiently high concentration that the plot of [LR] vs. [L] begins to "lay over". You may also see evidence of non-specific binding, which will have to be taken into account.

Tu,Mar.6

 

Ch. 10 in N&B, Ch. 11 in NB&B. This is one of the best treatments of the math and the methods used for doing ligand binding assays that you will find!

There is no protocol, you will have to design your own!

See notes ==>

Precision is the key to success in this experiment!

Lecture notes: ligand_binding.pdf

In this experiment you will have to make an intelligent guess (1< [L] < 10 uM) about the value for Kd (the paradox) since you will have to titrate the receptor with the ligand, getting some data points less than Kd, and going to sufficiently high concentration that the plot of [LR] vs. [L] begins to "lay over". You may also see evidence of non-specific binding, which will have to be taken into account.

Th,Mar.8

Competitive Ligand Binding: Determination of Kd for binding of a competitive ligand (either desthiobiotin or iminobiotin)

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In this part you will poise the system such that the [HABA:avidin] = [total avidin] as much as possible (i.e., 0.999[total avidin]) by the addition of an excess of HABA. Why do you want to do this?????

You will then begin titrating in your competitive ligand, monitoring the loss of absorbance due to HABA displacement using difference spectrscopy.

Turn in Ligand Binding report on Tuesday.

. Spring Break . .

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Tu,Mar.20

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

Ch. 7: Gen. Protein Purrification

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

Ch. 9: Sections 9.1, 9.2, and 9.3: AP purification.

Expt. 9-3 (NB&B). Day One: Stage 1 Enzyme

Lecture Notes: Protein_purification.pdf

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

Each day during the purification you will have to do an enzyme activity assay for that step in the process. Appendix 9-1 (p.242 in NB&B) describes assay methods in general, while 9-2 describes a Continuous Assay of AP.

You will also set aside an aliquot for performing SDS-PAGE gel and Bradford assays at the end of the purification (Apr.1).

Th,Mar22

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

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Day Two: Stage 2 and 3 Enzymes.

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Tu,Mar27

AP Purification: Stage 4 Enzyme by DEAE Chromatography. Ch.9 Day Three: Stage 4 Enzyme. .

Th,Mar29

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,Apr3

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,Apr5

Steady-State Kinetics: Competitive Inhibition by Inorganic Phosphate

Ch. 10: Sec. 10.5.

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.

Tu,Apr10

Th,Apr12

Tu,Apr17

Th,Apr19

Special Research Project

 

 

 

Too many to list

 

 

 

 

 

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

 

 

See Special Research Project page.
Tu,Apr

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,Apr24

No Class

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Th,Apr26

Research Presentations

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Tu,May 1

Research Presentations

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Th,May 3

.Turn in Final AP manuscript to TA

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The END!

 

Course Information | 463a Home


Biochemistry 463a
http://www.biochem.arizona.edu/classes/bioc463a/463a.html
Department of Biochemistry and Molecular Biophysics
The University of Arizona
jhazzard@email.arizona.edu 
All contents copyright © 1998-2000. All rights reserved.
Last revised Dec. 20, 2001