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Pease Note: For each lab session:
- Please thoroughly read the appropriate material in
Fundamental Approaches
for Biochemistry and Biotechnology by
Ninfa and Ballou (N&B).
- Check the Lecture and Laboratory Notes webpage for
additional information.
- Prepare a Flow Chart for each day's experiments
(see Course Information: Flow Chart).
- 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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Feb. 12
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Last day to drop without Drop/Add
form
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Mar. 11
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Last day to drop with a "W" using a Drop/Add
form
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Date
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Experiment
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Text
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Protocol
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Special Notes
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Th,Jan.17
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Introduction to BIOC 463a: Orientation, Course Objectives, and
Expectations.
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Please read Syllabus thoroughly.
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Lecture will meet in Shantz 247 for morning and afternoon classes.
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Tu,Jan.22
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Preparation and Characterization of Buffers
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Ch.1
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Exp_1.pdf |
Lecture Notes for Expt.
1.
See Lab Report.pdf for
instructions on writing you reports. Please have Tables filled
out prior to coming to class for all but the raw data.
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Th,Jan.24
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Buffers cont'd
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Tu, Jan.29
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Absorbance Spectra of PNPOH and PNPO-.
Detemination of the Extinction Coefficient for PNP.
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Ch.2
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Exp_2.pdf
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Lecture Notes for Expt. 2.
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Th,Jan.31
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Determination of the pKa of PNP in Phosphate Buffer and the Effect
of Alcohol on the pKa.
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Ch.2
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Exp_2.pdf
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Tu,Feb.5
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Colorimetric Determination of Protein Concentation: Lowry, BCA,
and Bradford Assays.
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Ch.3
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Exp_3.pdf
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Lecture Notes for Expt.
3.
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Th,Feb.7
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Determination of Protein Concentration: A280 Method and the Effect
of DNA on the A260/A280 ratio.
Determination of Protein Concentration: Heme Chromophore Methods
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Ch.3
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Exp_3.pdf
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Tu,Feb.12
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Size Exclusion and Ion Exchange Chromatography
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Ch.4
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Exp_4_1.pdf
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Lecture Notes
for Expt. 4
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Th,Feb.14
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Affinity Chromatography (Beta-galactosidase purification)
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Ch.4
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Expt. 4.2 (N&B).
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Tu,Feb.19
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SDS-PAGE
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Ch.5
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Expt_5.pdf
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.Lecture Notes for PAGE
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Th,Feb.21
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Native PAGE and Performing Enzyme Assays on a native gel.
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Ch.5
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Expt_5.pdf
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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.
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Tu,Feb.26
Th,Feb.28
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Mass Spectrometry and Proteomics
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Ch.5 (pps. 143-145). Please refer to your 462 textbook for
additional information
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All information will be online, see links on homepage.
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Class will begin at 9 am and 1 pm in BSW 243.
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Tu,Mar.4
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Western Transfer of Alkaline Phosphatase
Day 1: Running the SDS-PAGE gel and doing the Western Transfer.
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We will hand out the reading material for this procedure in class.
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Western Blots.pdf
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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.
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Th,Mar.6
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Western Transfer of Alkaline Phosphatase
Day 2: Binding of Antibody and performing the colorimetric assay.
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Tu,Mar.11
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Ligand Binding Equilibrium (Avidin and HABA) |
Ch.10 pps.
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Expt. 10-1.pdf and Expt.
10-1 Objectives 1,2, and 3.
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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 <= 0.2.
We will first do a titration of Avidin using HABA, from which
you can determine Kd.
Note: non-specific binding (N&B pps. 268-269) 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.
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Th,Mar.13
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Competitive Ligand Binding Assay:
Back Titration of HABA:Avidin complex with Biotin.
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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
(see N&B, pps. 260-261). The HABA:Avidin complex will the
be titrated with Biotin, from which it should be possible to determine
a value for Kd for Biotin.
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Tu,Mar.25
Th,Mar.27
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Molecular Biology Module, Isolation of the Cus F-YFP plasmid,
restriction digestion, and transformation of plasmid into competent
E. Coli cells
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Ch 11. in N&B for general information |
CusF-YFP expt.
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N&B has general information about routine molecular
biology procedures and techniques. We will provide specific details
about this expt prior to class. |
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Tu,Apr.1
Th,Apr.3
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Molecular Graphics meet in.
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
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Tu,Apr.8
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Alkaline Phosphatase (AP) Purification: Stage 1 Enzyme, Spheroplasts
and Activity Assays.
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Chs. 6 and 7.
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Expt. 7-1 (N&B). Day One: Stage 1 Enzyme |
Lecture Notes: Protein_purification.pdf
Make a copy of the Table on p. 172 prior to class.
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Th,Apr.10
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AP Purification: Stages 2 and 3 Enzyme by Heat and AmSO4
Precipitation.
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Ch.7
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Day Two: Stage 2 and 3 Enzymes.
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Tu,Apr.15
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AP Purification: Stage 4 Enzyme by DEAE Chromatography.
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Ch.7
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Day Three: Stage 4 Enzyme.
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Th,Apr.17
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AP Concentration Determination, Activity Assays, SDS-PAGE,
and Mass Spectrometry. |
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Review the material on Bradford Assays and pouring
SDS-PAGE (10%) gels. |
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Tu,Apr.22
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Steady-State Kinetics: Determination of Km, Vmax, and kcat of
AP
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Ch. 8
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Expt. 8-1.
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Lecture Notes: Steady_State_Kinetics.pdf
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Th,Apr.24
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Steady-State Kinetics: Competitive Inhibition by Inorganic Phosphate
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Ch. 8
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Expt. 8-2. The protocol given in N&B is for a Dixon method
of studying competitive inhibitors. Consult your BIOC 462B text
for a Lineweaver Burke method.
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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.
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Tu,Apr.29
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EDTA inhibition and using kinetic data to propose a mechanism
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A data set for inactivation of AP will be handed out for this
class, which you will analyze in class
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Th,May 1
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Research Presentations
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Tu,May 6
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Research Presentations
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