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	.
?	Last day to drop with a Deletion
?	Last day to drop with a "W"

Date	Experiment	Textbook Reading	Protocol	Special Notes
.	.	.	.	.
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	.
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.	.	.	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)	.	.	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	.	.	.
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.	.	Day Two: Stage 2 and 3 Enzymes.	.
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	.	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	.	.	Each group will give a short (<= 10 minute) presentation of the results from their project.
Tu,Apr24	No Class	.	.
Th,Apr26	Research Presentations	.	.	.
Tu,May 1	Research Presentations	.	.	.
Th,May 3	.Turn in Final AP manuscript to TA	.	.	.

The END!