A Word of Warning: Only download the material for the upcoming week's work; material for future dates are under revision.
Rooms and Times for Bioc 463a
- Section 1: Shantz 247@ 8 am for lecture
- Section 2: Shantz 247@ 1 pm for lecture
- Sections 1 & 2: Koffler 540 after lecture.
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Instructor |
Room and Phone Number |
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Office Hours |
Dr. James T. Hazzard (Course Coordinator)
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BSW 342A, 621-7118
Koffler 540, 621-9989
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Friday, 9 - 12 noon(Koffler 540, verify first with Dr. H.
(M, T, W, Th : usually can be found in Koffler 540). |
| Swapna Aravind |
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| Tiffany Mealman |
tba. |
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Kayla Polzin |
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Deboleena Sarkar |
tba |
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tba |
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Preceptor contact information is at the discretion of the individual
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By appt only |
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Preceptor contact information is at the discretion of the individual |
By appt only |
Molecular Graphics Websites
Instructions
for the JAVA/JMOL installation.
BIOC
462a JMOL scripts
NCBI
Home
NCBI/Structure
Home
VAST
Home
J. Biol. Chem. in the Classroom: Three papers from JBC related to Anfinsen's legendary work on bovine pancreatic ribonulcease that provides a historical and scientific framework for the Special Research Project:
RNase1.pdf: a JBC Classics article reviewing Anfinsen's work with RNase.
RNase2.pdf: an original 1960 JBC paper by Haber and Anfinsen about RNase.
RNase3.pdf: a very interesting JBC Classics article about the complete amino acid sequencing of RNase by William Stein and Stanford Moore important for Anfinsen's research.
Molecular Graphics Task Force has been established. If you would like to participate, contact Dr. H
Please read: A Fine-Structure Genetic and Chemical Study of the Enzyme E. coli AP.pdf by Garen and Levinthal. By now, you know quite a bit about this enzyme, therefore you should have a good idea what is really important in this paper and what can be skimmed over. For instance, they determine the molecular weight by ultracentifugation, how does this compare to the calculated molecular weight determined by ExPASy or the mass spectrum shown in class by Linda Breci (see Mass Spec notes below)? How does G&L's pI compare to the theoretical value from ExPASY. What does the activity vs. pH curve tell you, i.e. how many titratable groups are evident? How much specificity is there for the R- group for the various phosphorylated substrates say about substrate binding? There is also a very intersting discussion about the thermal stability of the enzyme. At this stage in your careers, these are the types of questions you should ask when reading a paper like this.
Please read: Structure and Mechanism of AP.pdf by Joseph Coleman. This is a review article; sections of importance have been marked with an "*", whereas sections of lesser importance with a "NO" (which does not stand for nitric oxide!). The mechanism at the end of the paper is based on the first, low resolution structure for AP. Using the mechansim for serine proteases as a reference, what very important step is missing from the mechanism for AP's catalytic reaction proposed by Coleman?
Please read: A Revised Mechanism for Alkaline Phosphatase.pdf by Stec et. al. This paper clarifies a key missing element in the Coleman paper.
Please read: Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Ion. Because of the complexity of this paper, we will discuss this paper on Tu, Nov. 15.
Special Research Project
General information for composing your final AP report: AP_Manuscript_Prep_f11.pdf
ap_massspec_data.pdf
Also, the grading rubric: AP_Final_Manuscript_RubricF11.pdf
Tu Nov. 29, Th Dec. 1, Tu Dec. 6: Research Lab Seminars
Final AP Manuscript is due on Dead Day, Dec. 8!!!!! |
Molecular Graphics Information
Jmol_workshop12.pdf
Pymol tutorials:
pymol_tutorial_stockwell.pdf
pymol_tutorial_rother.pdf
Miyashita's modified PyMol Exercise
Pymol1.pdf
Pymol2.pdf
Pymol3.pdf
Feb. 7: SDS-PAGE Exercise: Today, the primary goal is for everyone to learn how to pour, run, and analyze SDS-PAGE gels.
Lecture notes: PAGE.pdf
Protocols: expt_5.pdf
Feb. 9: Native Gel Enzymatic Assays:
In this exercise we will perform in-gel assays on two enzymes, E.coli b-galactosidase using the ONPG/Z buffer assay and E. coli alkaline phosphatase (AP) using a Fast Red Dye assay kit from Sigma as well as a commonly used AP substrate para-nitrophenyl phosphate (PNPP).
Jan. 31: Purification of Cytochrome c and Hemoglobin by Size Exclusion and Ion Exchange Chromatography.
Lecture notes: Column Chromatography.pdf
Protocol: Experiment 4.pdf
This homework assignment is to be turned in on Tuesday, Jan. 31: cytc_Hb_expasy_assignment.pdf. It is designed to prepare you for the column chromatography experiment.
Alert: It was brought to my attention that the ExPASy Protein Parameter function might be done early this week. As an alternative to using ExPASy to calculate the MW and pI parameters for the three proteins you might have to use another proteomic website called Protein Prospector. Please follow the instructions given in: ProteinProspector_backup.pdf
Feb. 2: Purification of E. coli beta-galactosidase by affinity column chromatography.
Please review the procedure for doing a Bradford assay prior to coming to class.
Lab Report and Scientific Writing Tips:
An example of a well written lab report can be found here: LabReport_Syna.pdf
An example of an actual manuscript submitted to Biochemistry can be found here: Effect of Viscosity on IET in Sulfite Oxidase
Instructions to Authors for the journal Biochemistry will be used for the remainder of the semester.
Lab_Report.pdf: General tips for composing ones thoughts prior to writing your lab report. Detailed information for what is to be included in the first few lab reports can be found at the end of the Protocol section along with supplimental questions that will also be turned in.
BMB_Sci_Writing.pdf: A general guide for scientific writing thoughtfully prepared by Prof. Marc Tischler.
manuscript_algorithm_bambed.pdf: An informative "How To" guide for writing and submitting a manuscript to a scientific journal AND for those suffering from writers block (from the journal BAMBEd).
The following links will be used as the semester progresses, do not down load until they are moved to the top pane:
Youtube video on pouring gels, check it out!: http://www.youtube.com/view_play_list?p=0DF3730B1D55C58B
AP related material
Include the interpretation of the mass spec data in the write up you turn in on Tuesday, no need to turn in seperately:
ap_structure_exercise.pdf.
AP_Structure_Exercise.pdf: To be turned in on Tuesday and included in your final AP manuscript.
End of the semester assignments:
edta_ap.pdf.
ExPASy homework !
ExPASy homework
Miscellaneous Material:
Molecular Graphic Workshop information: New_Jmol_Workshop.pdf
Files for PyMol:
Pymol1.pdf
Pymol2.pdf
Pymol3.pdf
The reason for learning this material are the questions that need to answered in writing your lab report for the Ligand Binding experiment: Molecular Graphics for Ligand Binding Experiment.pdf
REINCARNATION: a comical and metaphysical observation on a transformative process (with a Western flair).
Bioc 463 Assessment Excel File
Molecular Biology Homework
Mass Spec/Proteomics material:
http://www.chem.arizona.edu/facilities/msf/index.html
www.chem.arizona.edu/facilities/msf/BiochemLab/exercises.html
Go to the bottom left of the page
and click on the links there.
Mass Spec Homework: Here are the two files in a zipped folder, let me know if you have trouble opening either one.
New Mass Spec and Proteomics.zip
When you open the zipped folder, there should be a Word document and the .mgf file which contains the tandem mass spec data, save both to your computers . Let me know if there are any problems with opening these files.
Remember, you can always use PubMed and Wiki for this assignment.
Literature References for Mass Spec and Proteomics:
(1) Hanash, S.M. (2000) Electrophoresis 21 (6): 1202-1209: Biomedical
applications of two-dimensional electrophoresis using immobilized
pH gradients: Current status.
(2)Aebersold, R. and Mann, M. (2003)NATURE* 422 (6928): 198-207:
Mass spectrometry-based proteomics.
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