BCH 467 Analytical Biochemistry Laboratory, Exercises of Analytical Chemistry

Download BCH 467 Analytical Biochemistry Laboratory and more Exercises Analytical Chemistry in PDF only on Docsity! ARIZONA STATE UNIVERSITY GENERAL STUDIES COURSE PROPOSAL COVER FORM. Course information: Copy and paste current course information from Class Search/Course Catalog, Academic Unit Biochemistry Department _Chemistry & Biochemistry . Subject BCH Number 467 Title _ Analytical Biochemistry Laboratory Units: 3 Is this a cross-listed course? No If yes, please identify course(s) ee Is this a shared course? No If so, list all academic units offering this course Course description: Quantitative analysis, separation and purification of biological molecules. Applies chemical and physical methods to the characterization of biological macromolecules. Requested designation: Literacy and Critical Inquiry-L Note- a separate proposal is required for each designation requested Eligibility: Permanent numbered courses must have completed the university's review and approval process. For the rules governing approval of omnibus courses, contact the General Studies Program Office at (480) 965-0739. Area(s) proposed course will serve: A single course may be proposed for more than one core or awareness area. A course may satisfy a core area requirement and more than one awareness area requirements concurrently, but may not satisfy requirements in two core areas simultaneously, even if approved for those areas. With departmental consent, an approved General Studies course may be counted toward beth the General Studies requirement and the major program of study. Checklists for general studies designations: Complete and attach the appropriate checklist « — Literacy and Critical Inquiry core courses (L! Mathematics core courses (MA\ Computer/statistics/quantitative applications core courses (CS) Humanities, Fine Arts and Design core courses (HU. Social and Behavioral Sciences core courses (SB) Natural Sciences core courses (SO/SG Global Awareness courses (G) Historical Awareness courses (H. Cultural Diversity in the United States courses (C A complete proposal should include: {] Signed General Studies Program Course Proposal Cover Form & Criteria Checklist for the area Kd Course Syllabus XX] Table of Contents from the textbook and list of required readings/books Contact information: Name Scott Lefler Phone 7-6487 Mail code 1604 Email: — slefler@asu.cdu Department Chair/Director approval: (required) Chair/Director name (Typed}: Wilson Francisco Date: of ZL ap / Zoe Chair/Director (Signature): bison Srapnt2.w— Rev. 1/94, 4/95, 7/98, 4/00, 1/02, 10/08, 11/11/ 12/11, 712 Arizona State University Criteria Checklist for LITERACY AND CRITICAL INQUIRY - [L] Rationale and Objectives Literacy is here defined broadly as communicative competence in written and oral discourse. Critical inquiry involves the gathering, interpretation, and evaluation of evidence. Any field of university study may require unique critical skills which have little to do with language in the usual sense (words), but the analysis of spoken and written evidence pervades university study and everyday life. Thus, the General Studies requirements assume that all undergraduates should develop the ability to reason critically and communicate using the medium of language. The requirement in Literacy and Critical Inquiry presumes, first, that training in literacy and critical inquiry must be sustained beyond traditional First Year English in order to create a habitual skill in every student; and, second, that the skills become more expert, as well as more secure, as the student learns challenging subject matter. Thus, the Literacy and Critical Inquiry requirement stipulates two courses beyond First Year English. Most lower-level [L] courses are devoted primarily to the further development of critical skills in reading, writing, listening, speaking, or analysis of discourse. Upper-division [L] courses generally are courses in a particular discipline into which writing and critical thinking have been fully integrated as means of learning the content and, in most cases, demonstrating that it has been learned. Students must complete six credit hours from courses designated as [L], at least three credit hours of which must be chosen from approved upper-division courses, preferably in their major. Students must have completed ENG 101, 107, or 105 to take an [L] course. Notes: 1. ENG 101, 107 or ENG 105 must be prerequisites 2. Honors theses, XXX 493 meet [L] requirements 3. The list of criteria that must be satisfied for designation as a Literacy and Critical Inquiry [L] course is presented on the following page. This list will help you determine whether the current version of your course meets all of these requirements. If you decide to apply, please attach a current syllabus, or handouts, or other documentation that will provide sufficient information for the General Studies Council to make an informed decision regarding the status of your proposal. Literacy and Critical Inquiry [L] Page 4 Course Prefix Number Title Designation BCH 467 Analytical Biochemistry Laboratory L Explain in detail which student activities correspond to the specific designation criteria. Please use the following organizer to explain how the criteria are being met. Criteria (from checksheet) How course meets spirit (contextualize specific examples in next column) Please provide detailed evidence of how course meets criteria (i.e., where in syllabus) 1 and 3 There are 1350 points possible in the course with 1100 (81%) of those points derived from six written lab reports See page 2 of the syllabus, marked C-1 and C-3 2 Lab reports are written in a style very similar in format to typical articles from a scientific journal. The Introduction section relates the students' work to previously published material. The Materials and Methods section describes the procedure used to gather evidence. The Results section translates the raw data into a meaningful interpretation, putting the data into the context of previously published material. The Discussion section requires the student to critically think about the results and the validity of the information that they are presenting. See page 3 of syllabus and the entire page titled Lab Reports for BCH 467, both marked C-2 4 For the first 4 of 6 lab reports, all reports are returned within one week of submission, allowing time for students to see grading comments prior to submission of the next report. See page 3 of the syllabus, marked C-4 BCH 467 - Analytical Biochemistry Laboratory Course descri biological macromolecules. Enrollment requirements: Pre/Co-requisite: BCH 462 with C or better if completed Units: 3 Repeatabie for credit: No General Studies: L Offered by: College of Liberal Arts and Sciences Class meeting details Class #: Days: Start: End: Location: 19414 w 730 AM 8:20AM Additional class details: Component; Recitation Session: Session © Required components: Recitation, Laboratory Dates: 1/13/2014 - 5/2/2014 Last day to enroll: January 19, 2014 Drop deadline: January 19, 2014 Course withdrawal deadline: April 06, 2014 Instruction Mode: In-Person Fees: $100 class fee Books: lf View books for this class Tempe -PSH152 Seats open: 5 of 1758 Spring 2014 ion: Quantitative analysis, separation and purification of biological molecules. Applies chemical and physical methods to the characterization of BCH467: General Biochemistry Lab Spring 2014 Instructor: Dr. Scott R. Lefler Phone: 727-8282 Office Hours in PSH-237: see Blackboard Contacts Email: slefler@asu.edu Include BCH 467 in the subject line! Also available by appointment. Please email or call with the specific day and time you would like to meet. Location: Recitations will be online, labs in PSH-431 or PSH-430. Lab section Teaching Assistant email Mon 1:00 Dayna Peterson Dayna.S.Peterson@asu.edu Tues 8:00 Jason Maxwell jdmaxwe1@asu.edu Tues 11:00 Jesse Coe jdcoe@asu.edu Tues 1:00 Andrew Serban ajserban@asu.edu Wed 12:00 Wei Feng wfeng13@asu.edu Wed 2:30 Nicholas Sisco nicksisco@asu.edu Thurs 8:00 Pankti Shah phshah8@asu.edu Thurs 12:00 Andrey Kanygin akanygin@asu.edu Thurs 2:30 Eduardo Espiritu eespiri1@asu.edu Fri 8:00 Anshu Sharma asharm66@asu.edu Fri 11:30 Jacob Hilton Jacob.K.Hilton@asu.edu Text: Fundamental Laboratory Approaches for Biochemistry and Biotechnology (2009). Ninfa and Ballou; John Wiley & Sons. Either the first (ISBN 1891786008) or second (ISBN 0470087668) edition will suffice; I will post reading assignments for both editions. Required for lab each day: Lab notebook (a bound composition notebook is fine) with numbered pages, a USB drive, a fine-tip permanent marker pen, safety goggles and lab coat. The computers in lab are not connected to the internet so the USB drive is absolutely required on days where data is collected on the computer. For that reason, it is best to bring a USB drive every day. Course Description: This is a biochemistry laboratory course designed to introduce the student to basic techniques commonly used in protein and DNA work. Emphasis is placed on DNA manipulations and protein expression, purification and analysis. A schedule of labs can be found on the last page of the syllabus. Course website: Lab protocols and other important information will be available on myASU/blackboard. Please make sure that your email address is updated in the event that messages are sent from the site. You must print a copy of the lab protocol for use during lab. It is recommended that you read over the protocol prior to lab. BCH467: General Biochemistry Lab Spring 2014 If you fail to submit all six lab reports to SafeAssign on time, you will not receive a passing grade in the course. We will not reopen SafeAssign assignments, this is your only warning. Email contact: I welcome email contact; however, please keep the following items in mind. Please use email only if the expected response is brief. If the topic in question requires a discussion, please see me in person or call. I will not answer questions that can be clearly answered by consulting the syllabus. When emailing me, keep in mind that I have other courses so you must include BCH 467 in the subject line. Finally, correct spelling, capitalization and punctuation is required. If I cannot understand your email, I will not be able to respond. Syllabus contract: You must sign the syllabus contract stating that you have read the syllabus and agree to the conditions set forth in the syllabus. The syllabus contract must be submitted to your TA no later than the second lab period. Course Withdrawal: Please discuss any plans to withdraw from the course with me. I would rather work with you (such as granting an incomplete if warranted) than to require you to repeat the course. Requests for a grade of incomplete must be made during the semester as I will not change grades after the completion of the semester. BCH467: General Biochemistry Lab Spring 2014 Lab Schedule Week Lab # Title Report Jan 13-17 Introduction (Monday lab) None Jan 20-24 Introduction (all other labs) None Jan 27-31 1 Restriction Mapping 1 Feb 3-7 2 PCR and DNA Sequencing 2 Feb 10-14 3 DNA Restriction and Ligation Feb 17-21 4 Transformation & Expression in Liquid Culture Feb 24-28 5 Expression on Solid Media 3 Mar 3-7 6 Protein Affinity Purification Mar 10-14 Spring Break Mar 17-21 7 His tag Cleavage & DEAE Chromatography Mar 24-28 8 SDS-PAGE 4 Mar 31 - Apr 4 9 Protein Assays Apr 7-11 10 Chromophore Optical Properties and pKa 5 Apr 14-18 11 Enzyme Kinetics 6 Lab report information: All lab reports are due at the start of your lab period. If your report is submitted more than 5 minutes after the start of lab, it will be considered 1 day late. Report # Due Date Page limit Points 1 Feb 3-7 10 100 2 Feb 17-21 13 100 3 Mar 3-7 17 300 4 Mar 31 - Apr 4 23 300 5 Apr 14-18 13 200 6 Apr 21-25 10 100 Lab Reports for BCH467 Lab reports must by typed with page numbers including page count (e.g. 3 of 15). Any equations and math should be typed using a program such as Mathtype or Microsoft Equation Editor. Figures must be scanned and pasted directly into the text. They should be numbered, clearly labeled and must be referenced in the text. You must adhere to the page limits—any text beyond the page limit will not be read. LAB REPORTS MUST BE TYPED IN 12 POINT FONT AND DOUBLE SPACED! Sections of the Lab Report: Title (you do not need a title page) Abstract (10%) 1. Brief project description 2. Most important results and conclusions Introduction (25%) 1. Objective or purpose of the experiment (what are the goals?) 2. Brief discussion of theory behind the experiments and techniques 3. Any chemical or biochemical reactions 4. Any equations used for analysis of data Materials and Methods (15%) 1. A narrative of what you did in the lab. In order to write this section, you need to read a journal article (Journal of Biological Chemistry is a great example). 2. Should include all reagents and instruments used in the experiment. 3. Should enable the reader to repeat your experiment and arrive at similar results 4. Should be clear and concise 5. DO NOT INCLUDE A LIST OR TABLE OF MATERIALS! Results (20%) 1. An opening statement to describe the general results obtained 2. Raw data (tables, graphs, photos of gels, etc.) in an organized manner and clearly labeled—figures must be scanned and pasted directly into the text 3. Graphs must be generated with a program such as Excel or Sigma Plot 4. Calculations (refer back to equations mentioned in introduction) 5. Factual description of results 6. Figures and tables must have a title and description Discussion (30%) 1. Significance of results 2. Discussion of unexpected results, problems encountered, failure of experiment 3. Interpretations supported by data 4. Limitations of data 5. Suggestions for improvement 6. Questions from text References (may be single spaced) 1. Citations from text 2. Other literature While there is no grade for this section, lack of references will impact the introduction section. Ce 2.8 Appendix Calculating Titration Curves for Benet Acids and Other Multiple Binding Site Receptors... serseseneesasdsensay snveseeserdabetiaaavannfeasstO2 2.9 Equipment Used in This Course.......c.ccscsccssesssesscasccssussees cesseaneeneesseessesseee OF CHAPTER 3: SPECTROSCOPIC METHODS 65 3.1 Introduction sessccossssesenassccsioncsnsacananconenvensnncooscnennosnowonssnnesnxonnnesenanrerannonnnens 65 | 3.2 Design and Properties of Spectrophotometers.. | 3.3. Effects of Spectral Bandpass and Stray Light ........cccccseeseseeesseeesseeneeseee 72 3.4 Recording Spectrophotometers. ..........csssseseseseseeceesseseseetssseaseeteeereesessseneee 74 3.5 Fluorescence Spectroscopy.....cccsccesescssseseseseseressessssssseseseseeeeeneseneneeeeeene 15 3.6 Chromogenic and Fluorogenic Reactions Used for Analysis... 83 3.7 Other Spectroscopic Techniques ..........cccsccseseeseessesssesssssesesteeseneeeseeseeenses 84 3.8 Mass Spectrometry (MS)... sccsesesesessssseeneseesesssssessassssceseseseensnsesssneesee 94 Experiments 3-1 t0 3-4 .sccssessacrescevasusnosensissasenrenenansenenneonsvesndecowenavectoroneastestanseee 99 Reagents Needed for Chapter 3 CHAPTER 4: QUANTIFICATION OF PROTEIN CONCENTRATION 105 4.1 Purposes of Protein Quantification ........c.ccccsssessssessseseseseseeeneeessneeenenenes 105 4.2 Factors to Consider in Choosing an ASSay.......ccsccsessesesssesseseresseseseasenenes 107 4.3 Non-Colorimetric Procedures for Quantification of Proteins... 108 4.4 Colorimetric Procedures for Quantification of Proteins ............:.:ceseeeee 110 | Experiment 4-1. Reagents Needed for Chapter 4.0... ccccsssesesesesesetsseseeeesesesrscsesessseneesesensenen 119 viii CHAPTER 5: CHROMATOGRAPHY 121 3.1 Introduction saisieicrnausennnsarnnsauncnwinnanmaamnnatesnmmess 121 5.2. Gel-Filtration (Size Exclusion or Gel-Permeation) Chromatography ....... 125 5.3 Affinity Chromatography .. 5.4 Ton-Exchange Chromatography .......cccccecsesesssseseseseseeeeseseneneseessasseseerer 143 5.5 Hydrophobic Interaction Chromatography........cccesscsseeeeeeeessesessseseseeeee 149 Experiments 5-1 and 5-2..............-smqsuguvssousyassavesgassuaneasnadeguaeabened aajaetaneseaseteas 152 Reagents Needed for Chapter 5 ......c.ccecsesssssesseeeeeseeeeeeeecsesesseeseseseataceenseseeneeeee 160 CHAPTER 6: GEL ELECTROPHORESIS OF PROTEINS 161 6.1 Process of Electrophoresis.......cccccsscsesesesesessssseseseseseesetserenesssesesesseeeee 161 6.2 Polyacrylamide Gels sssssjssicsissscisssssesnvaerageatsarsesnodsnsycarncessonsseacdsetusessenvevee 163 6.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins........ 165 6.4 Detection of Proteins in SDS-Polyacrylamide Gels ..........ccceeeeeeee 171 6.5 Applications of SDS-PAGE ..........ccssesessssesessssssssesssnessssstavessessnssesesesnees 173 Experiments 6-1 and 6-2......cccccssssssesssesseeesesteescsnesesesseneseseenenesesseneseseaneneseeens 183 Reagents Needed for Chapter 6 CHAPTER 7: OVERVIEW OF PROTEIN PURIFICATION 191 | FAA Untroduction ......cccscscssesssseessesessseseenesseeeseassvanessanenenessanensseereneseesaranensaetes 191 7.2 Development of a Suitable Assay Procedure .........csceseceesseesseeeeeeseeee 193 7.3 Time, Temperature, and Yield.. 7.4 Selection of the Best Source Material.......c...ccceecseseseseeeseeeseseseeeeeeee 195 7.5 Solubilization of the Protein ........ccccsssesssseessecesseesseesseaeeeeseereeseneseeenees 198  7.6 Initial Steps of Purification... 7.7 Developing a Series of High-Resolution Chromatographic Steps............. 201 7.8 Methods Used to Change Buffer and Concentrate Protein Samples ......... 204 7.9 A Logical Series of Steps 7.10 Storage of the Purified Proteit .......cscsscscccsssssssssssssssssssssssssssssssssssssssssses 205 TAL The Protein Purification Table.........:cscsssssssssssssssssssussssssssssesssesssssssssse 206 CHAPTER 8: SUBCELLULAR FRACTIONATION 209 8.1 Introduction, +209 8.2 Structural Organization of Prokaryotic and Eukaryotic Cells.....ccccsss0..< 210 8.3 Overview of Fractionation PROLOCOLS...eesesccssseessessccessentessescesssneeteeseensme dh 3 Experiment 8-1 Reagents Needed for Chapter Bos ccseressccccsnsssneescsensssuneeseesssssesesstesssssesesssssssaseeeeesn 227 CHAPTER 9: ISOLATION AND CHARACTERIZATION OF AN ENZYME—ALKALINE PHOSPHATASE FROM ESCHERICHIA COLI 229 DL Objectives... ecssssesssssssssssssssesesssseceesusssessessssseessusssassessassssssuassssssees 229 9.2 Introduction and Basic Principles... 9.3. Purification of Alkaline Phosphatase 9.4 Characterization of Purified Alkaline Phosphatase ...0.....cccccscsssssseeseeseees 242 Appendix 9-1 Assay of Alkaline Phosphatase .., Reagents and Equipment Needed for Chapter 90.0... cecsesssessecssessseaneeseeseesees 247  14,5 Legal and Bthical Issues ...........ccscsssssescssssessssssesesscscsssscorsesssetessiveceesneeense 410 Experiments 14-1 t0 14-6....c.csccssssscsssssssssssssssessssesssussssessssosssssavensvecivecsvecenecsuese 412 CHAPTER 15: USING THE COMPUTER AND THE INTERNET FOR BIOCHEMICAL RESEARCH AND COMMUNICATION 425 15.1 Introduction to the World Wide Web (the Internet) ..........ccssccsessesssseesseess 425 15.2 Information on the Internet Useful to Biochemists............cc.cssessesssseessseess 431 15.3 Literature and Data Searches chinese aeeaseasncensoneesenonuancecianesuosseneans 440 15.5 Other Useful Information ........c.ccccssecsssessssessssesssecssecssscsssessuccsucessscesssssave 442 INDEX 447 xiii List of required readings From Ninfa and Ballou, Fundamental Laboratory Approaches for Biochemistry and Biotechnology: Chapter 2-8, 10-11, 13-14 From Lehninger’s Principles of Biochemistry, 5th edition (this is the required textbook used in the corequisite course, BCH 462): Chapters 6.3, 9 From Methods in Enzymology: Volume 152, pages 61-87 and 113-129 Volume 155, pages 335-350 Volume 204, pages 63-113 Volume 218, pages 104-121 and 154-172 In addition to the list of readings, I have worked with the chemistry librarian (previously Olivia Sparks, now Linda Shackle) to develop two separate tutorials for evaluation of information sources. This has helped students choose quality information sources as background material for writing their lab reports. Since first implementing this tutorial, we have seen a dramatic drop in the use of random websites as information sources, replaced largely by peer-reviewed literature or textbook sources.