Elemental Analysis - University of Warwick：元素分析-华威大学.doc

_ MOAC DOCTORAL TRAINING CENTRE NOVEMBER/DECEMBER 2012 CH921: TECHNIQUES FOR THE CHARACTERISATION OF BIOMOLECULES COURSE LEADER: DR. ANN DIXON Contributors: Prof. Alison Rodger; Prof. Peter OConnor; Prof. Steven Brown; Dr. Claudia Blindauer; Dr. Vilmos Fulop; Dr. Jozef Lewandowski, Dr. Ivan Prokes 2 TABLE OF CONTENTS _ PAGE Deadlines and other important information3 Timetable4 Essay assignment5 Laboratory and workshop manual: Workshop 16-7 Protein List8 Workshop II9 Aims and Assessment10 Guidelines for preparation of lab report 11 Experiment I12-13 Experiment II14-18 Experiment III19 3 The following contains important information regarding deadlines, submission of work, etc. Please read CAREFULLY. ATTENDANCE AND SUBMISSION OF WORK _ Attendance at all scheduled sessions will be mandatory and recorded. Please ensure Sarah Shute has your correct email address. You will be notified of timetable changes by email with at least 24 hours notice. Failure to note timetable changes will result in loss of credit for attendance. As:MIT students to submit all work to Christina Forbes. MOAC students to submit all work to Sarah Shute. Plagiarism policy: Any text directly cut and pasted from the internet or any online or electronic source will be automatically regarded as plagiarism. In cases where a particular phrase is reproduced directly from a published source (of any type), then the source should be referenced in full at the point at which it is quoted. Furthermore, the amount of directly reproduced phrases should be minimal and limited to what is essential to support the arguments presented in the text. In any case the total amount of directly reproduced (and referenced) phrases should not exceed 5% of the full piece of work. Complex diagrams, which would otherwise be difficult to reproduce, may be taken from a published source provided that the source is directly referenced and the appropriate reproduction permission has been achieved, if required (not needed for essays or laboratory reports). ASSESSED WORK AND DEADLINES _ Deadlines are serious. Marks will be deducted from late work (1% / hour late; 5%/day late) unless an extension is granted in writing from Dr. A. Dixon. Workshop problems/proof of completion: Due by 5 pm on the day of the workshop. Essay:Due Tuesday 20 November at 4 pm. NMR assessment:Due Tuesday 4 December at 4 pm. Laboratory reports:Due Tuesday 11 December by 4 pm. An oral examination (with 2 hours written/reading work before hand, to be submitted at the oral) will take place on Monday 3 December. You may take up to one A4 sheet of handwritten notes only into the written part of the examinations. BREAKDOWN OF MARKS _ Assessed work: 45% Exam: 45% Attendance: 5% Laboratory conduct: 5% 4 CH921 TIMETABLE AUTUMN 2012 _ WEEK 6: NOV 5 AND 6 Mon. 9:00-10:00: Introduction to techniques for the characterization of biomolecules (Dixon, MOAC Lecture Room) 10:00-12:00: Databases workshop (Dixon, MOAC Lecture Room) 13:00-17:00: Obtain Essay title and begin work on Essay, perform calculations required for Experiment I Tues. 9:00-11:00: UV lecture and workshop (Rodger, MOAC Lecture Room) 11:00-13:00: Fluorescence lecture, until 6 pm) Tues. NMR Demonstrations 9:30-12:30 (MOAC Students): (Lewandowski, Milburn House) 10:00-12:00 (As:MIT Students): (Prokes, Milburn House) WEEK 10: DEC 3 AND 4 Mon. 9:00-17:00: EXAM Tues. 10:00-11:00: Pre-lab (Dixon, MOAC Lecture Room) 11:00-16:00: Laboratory: Exp. V (Dixon, Chemistry B309) *Deadline 2: NMR Assessment due (4 pm) WEEK 10+1: DEC 11 Tues. *Deadline 3: Lab reports the UniProt Reference Clusters (UniRef); and the UniProt Archive (UniParc). UniProt is a collaboration between the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR) with the objective of the curation and organisation of huge numbers of protein sequences. UniProt gives information on the function(s) of the protein; protein-translational modification(s); domains and sites; secondary and quarternary structures; similarities to other proteins; sequence conflicts and variants; and disease(s) associated with deficiency(s) in the protein. There is a high level of integration with other biomolecular databases. To enter UniProt, use the following website link to the Sequence Retrieval System homepage: / Select “UniProt KB” (UniProt knowledge base) from the “Search in” drop down menu, but note that there are other options available that you can explore in your own time. In the “Query” box, type the name of a protein or other keyword. As a practice run, type “Ubiquitin”, then click “Search”. You should have about 85,000 hits thats a lot of information about Ubiquitin. But youll need to narrow it down as you will most often be looking for information about a particular protein. Click the “advanced search” link on the right-hand side of the screen, and type in another search term to narrow down the number of hits. In this case, we will search for Ubiquitin from a particular organism. Select “Organism” from the “Field” drop down menu, but again note that there are other options available that you could use to narrow your search. Then type “Human”, select from the list, and click “search”. Now you should have over 3,000 hits that is still a lot, but in this case the one you want is the first hit (primary accession number P0CG47, UBB_HUMAN). Note: The primary accession number is a number that will not change and is used for only one protein, and this can be transferred to different databases. Select UBB_HUMAN by clicking on the accession number (P0CG47), and this will bring up the file on this particular protein. Scroll down and you will see a large variety of information about this protein, including key references, links and the protein sequence. Go to the protein sequence and you will see a drop down menu labelled “Tools”. You will now use these tools to analyse the protein sequence. PROTPARAM _ To obtain data on chemical and physical properties for a given protein found using UniProt, a tool called ProtParam can be used. This can be accessed by clicking “ProtParam” in the “Tools” drop down menu next to the protein sequence, and then clicking “go”. Alternatively, you can go directly to the ProtParam analysis website at: 7 /tools/protparam.html and entering either the primary accession number or the sequence of the corresponding protein. Once you have clicked “ProtParam” in the “Tools” drop down menu next to your protein sequence, select the region that is the main chain (1-76 in the case of Ubiquitin). This will give information about the number of amino acids; molecular weight; amino acid composition; chemical formula and extinction coefficient with and without disulfide bonds amongst other information. RCSB PROTEIN DATA BANK _ For information on the structure of a protein use the Protein Data Bank, web link: /pdb/home/home.do You can search this database using the primary accession number by clicking the “Advanced Search” link on the upper right hand side of the screen. In the Advanced Search interface, select “UniProtKB Accession Number(s)” in the “Choose a query type” drop down menu, and enter your accession number in the “Accession IDs” box. Then select “Submit query”. You should get approximately 10 hits. To start, you should look for structures (a) of just the protein (no bound ligands, etc), and (b) with the highest resolution. For this example search, you could select the structures with the PDB IDs 2XK5, 3NOB, or 2XEW. This will connect you to a page with several thumbnails at the top where you can see structure and sequence details as well as many other types of information. Have a look around, then view the structure using the .jmol viewer. You can save an image of the structure by right clicking and selecting the appropriate option for the file type you prefer. You can also download the structure file by selecting “Download Files”, and you can display the complete file in text format. With less well known proteins it is important to search for any missing residues. To do this use find under edit on the main toolbar and search for MISSING. YOUR ASSIGNMENT _ You will be assigned a protein from the list on the following page. Search for and find the protein you have been assigned in the UniProt database as you just did above. In a separate document, youll need to include the following: A. UniProt entry for that protein, including the protein sequence B. ProtParam analysis output for the protein C. An image of the X-Ray or NMR structure for that protein When you are done, youll need to show your report to a demonstrator in order to be approved. This is a pass / fail workshop, and you will need to perform the exercises until you pass. You will use the information you collect in this workshop to help you complete the lab 8 tasks (you will be using the same protein you were assigned here to perform your lab experiments), so youll need to bring a copy of your report to the lab. PROTEIN LIST _ Lysozyme Ribonuclease A -Lactalbumin Myoglobin -Chymotrypsin Cytochrome c 9 WORKSHOP II: FITTING OF CIRCULAR DICHROISM DATA DICHROWEB _ Dichroweb is an online circular dichroism analysis facility. To access Dichroweb for analysis of CD data, use the following website link and then follow the instructions below: www.cryst.bbk.ac.uk/cdweb Select START ANALYSIS Login (you will be given a password for this program at the start of the module) Enter file information. The file format is Jasco 1.50 if using data as a .txt file directly from the CD machine (this will be in units of mdeg). If the data has been converted to units of delta epsilon, use the free format (the answer you get ought to be the same whichever units you use). The input units are machine units. Analysis programs - SELCON and CONTIN may be the best choice. Reference set use the best to fit your data. Output format default, and output units - machine units. Submit the form. Protein concentration can be calculated using (found in the protein databases workshop) in conjunction with the A280 and Beers Law. Mean residue weight can also be calculated using the molecular weight and number of residues. YOUR ASSIGNMENT FOR EACH PROTEIN _ From the protein databases, you should determine the extinction coefficient, molecular weight, number of residues, and the protein sequence. Use dichroweb to generate plots using ONE fitting program e.g. SELCON. In MS Excel, plot your original experimental data in machine units and as a delta epsilon plot (in terms of amino acids). Also plot dichroweb experimental and fitted data. Compare your experimental data with the dichroweb data. Record the percentage secondary structure of your protein. * Note your username and password here (there will be provided to you): Username: _ Password: _ * Practice data set (monoclonal antibody fragment) available on CH921 website: 10 LABORATORY PRACTICALS INTRODUCTION _ The aims of this lab course are to familiarize you with biological samples and teach you competence in a set of standard spectroscopic and analytical techniques. These include: COSHH risk assessment, reagent preparation, and protein sample preparation (Experiment I). Protein concentration determination (Experiment II). Protein secondary structure determination by circular dichroism (Experiment III). You will be working in pairs in lab, but you will be responsible for independently writing up your lab reports. Important: Please read the relevant lab scripts BEFORE coming to lab - you must be prepared in order to complete experiments in the allocated time. No extra make up session will be available. ASSESSMENT _ Hand in a brief description of the experiments you performed (enough detail so you could look up your notes during your project and use the techniques), plots of the spectra you have recorded and the structural deductions you can make from them. Demonstrators will also be giving you a grade for laboratory work. Aspects being assessed will include: Improvement of laboratory skills with respect to sample handling Tidiness and cleanliness Accuracy of results Care of equipment Organization and efficiency in the laboratory. Your laboratory reports may form the basis of part of your oral examination for this module. Also include information from the databases about the protein you have worked with including: molecular weight, amino acid residue content, extinction coefficient, -helix content as determined from the crystal structure. BEFORE COMING TO LAB _ Read lab scripts for the current day. Perform all required calculations - lab time is limited and you will not be allowed to stay longer than scheduled session. Obtain lab coat, safety glasses and lab book and bring ALL THREE to every lab session. If you do not have all of these items as well as your lab manual, you will be asked to go get these items. 11 FORMAT FOR LAB WRITEUPS _ Write up Experiments 1-3 as a single report. Reports should be NO LONGER than 15 pages with 1.5 spacing between lines and no smaller than 11 point Arial font. Points will be deducted for longer reports/smaller font. You will be marked on scientific content as well as use of correct spelling, grammar, formatting and clarity. Remember - someone has to read this! I. Background/Introduction: Shows fundamental understanding of why we did the experiment, how the experiment works, and what the advantages/disadvantages are. Purpose of the experiment. Important background and/or theory. Description of specialised equipment. Justification of experiments importance. II. Methods Briefly describe procedure and any modifications to procedure (dont rewrite lab script). III. Experimental Results Present data in tables and graphs (dont forget to label all axes, number figures, and provide titles). Refer to all tables and graphs in the text - i.e. use complete sentences to draw attention to key points in tables or graphs. Provide a sample calculation for each type of calculation. This is the only way to get partial credit for incorrect calculations. Also, for each calculation highlight the final answer in bold so I dont have to hunt around (and possibly not find) your answer. State key results in sentence form, and summarize results in tables. All figures must contain figure captions. IV. Discussion This is the most important part of the report, where you can show your understanding of the results of the experiments. Discuss the significance or meaning of the results. Analyse and interpret results and analyse experimental error. Answer questions posed in lab. V. Conclusion Very brief - did the experiment work and what did you learn? 12 EXPERIMENT I: REAGENT AND PROTEIN SAMPLE PREPARATION REAGENT PREPARATION _ You will be preparing a buffer solution and using it to prepare your US protein stock solution and all subsequent dilutions of both US and SS. BUFFER PREPARATION _ The following table (Table 1) describes how an important buffer, phosphate buffer, is prepared at a range of pH values by mixing different volumes of two stock solutions. (From Methods in Enzymology, Vol. 1, p.138) Use Table 1 to prepare 100 mL of 50 mM phosphate buffer, pH=7. Phosphate buffer. Stock solutions:A: 0.2 M solution of monobasic sodium phosphate (27.8 g in 1000 mL H2O). B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4.7H2O or 71.7 g of Na2HPO4.12H2O in 1000 mL H2O). (mL A + mL B, diluted to a total of 200 mL) Table 1: A (mL)B (mL)pHA (mL)B (mL)pH 45.055.06.9 92.08.05.839.061.07.0 90.010.05.933.067.07.1 87.712.36.028.072.07.2 85.015.06.123.077.07.3 81.581.07.4 77.522.56.316.084.07.5 73.526.56.413.087.07.6 68.531.56.510.590.57.7 62.591.57.8 56.593.07.9 51.049.06.85.394.78.0 PROTEIN SAMPLE PREPARATION _ You will now make up the protein samples which you will use in Experiments 2 and 3. You will again be assigned the protein you used in Workshop I (Intro. to Protein Databases). Before weighing out your protein, you must have read ahead in the lab script and calculated the amounts of these substances you will need. 13 You will be making up your protein sample to be approximately 1 mg/mL by preparing a sample of known weight and volume. However, this will not be an accurate guide to concentration, as your protein may not be pure from the manufacturer and may be contaminated with salts or nucle