2014 Insert Schedule Here Your ScROLL website http://eppingcache.net/LQB681ScROLL/681.html OBJECTIVES of this unique unit Each student will be assigned an enzyme and contribute to the design of a method for purifying and assaying the enzyme activity. In this Unit you will: discover the biochemistry section of the library, learn how to ORGANISE and PLAN an experiment, learn how to follow a recipe and prepare solutions, set up and perform your own biochemical assays, use several procedures to separate proteins, experience the “team approach” to modern bioscience research, encounter setbacks and learn to deal with them, and construct a balance sheet for the recovery of protein and enzyme activity. In this unit you can and will make mistakes and learn to rectify mistakes without penalty. Some VERY IMPORTANT notes DO NOT REMOVE RELEVANT TEXTS FROM THE LIBRARY. THEY MUST BE AVAILABLE FOR USE BY ALL OTHER STUDENTS IN THE CLASS! A booking sheet for the High Speed centrifuge will be posted on level 9. The timing of the spins MUST BE STRICTLY FOLLOWED. Both the Booking Sheet AND your protocol MUST specify 3 things for EACH operation: the rpm, the gav AND the rotor type for EVERY SPIN. ALL EQUIPMENT (COLUMNS, BEAKERS, etc.) CARRIED OVER FROM ONE PRACTICAL SESSION TO THE NEXT MUST BE CLEARLY LABELLED WITH: The group name AND the PRECISE contents (e.g. "DEAE Sephadex A50", NOT "SEPHADEX") OR IT WILL BE THROWN OUT! DO NOT REMOVE any electrical equipment from the cold room. Condensation will form and this may short-circuit the device! A list of reagents must be lodged online before 10.00 a.m. on the FRIDAY morning prior to the Monday prac class or your group may be penalised! If there is a possibility that something could go wrong at a particular step, DO NOT USE ALL OF YOUR SAMPLE FOR THAT STEP!!! e.g. If you make a mistake during a chromatographic separation, you can repeat the step provided you still have material in reserve. At the end of the semester, you will be required to submit a BALANCE SHEET that accounts for the recovery of enzyme activity at a number of stages during the purification procedure. This must be included in a report that summarises the semester's practical work. AT VARIOUS STAGES THROUGHOUT THE ISOLATION PROCEDURE YOU MUST FREEZE A SMALL ALIQUOT OF MATERIAL FOR PROTEIN AND ENZYME ACTIVITY ANALYSIS TO ENABLE THE CONSTRUCTION OF THE BALANCE SHEET FOR ENZYME RECOVERY. Saving duplicate samples at 4C also may be of advantage if your enzyme is cold-labile. Students using yeast cells must dry the cells for several days. Weigh out approx 15% of the amount of material required to take into account any potential water loss. Textbooks & References There is no prescribed textbook for LQB681 HOWEVER these excellent texts are highly recommended for students planning to continue in the field of protein purification. Protein Purification. P.L.R Bonner, ed. (2007, 1s edn). Taylor & Francis, Publ. ISBN 978-0415-38511-4 Protein Purification. R.K. Scopes, ed. (1994, 3rd edn). Springer-Verlag, Publ. ISBN 3-54094072-3 Modern Experimental Biochemistry. R. F. Boyer, ed. (1993 2nd edn). Benjamin Cummings ISBN 0-8053-0545-9 Your Weekly Reagents List To enable the preparative staff to have the appropriate reagents available to students on the required day, it is important that they be provided with a list of REAGENTS and EQUIPMENT as soon as possible. The list for those groups commencing in week 5 MUST be submitted before 10:00 am Friday 15 August 2014 and should adopt the following format:Date Aug 18 Reagent MgSO4 sucrose/HEPES (pH 7.4) Volume 250 mL 500 mL Molarity 50 mmol/L 250 mmol/L Equipment chilled centrifuge 50 mL buckets x 6 pH meter Your Timing When you plan your detailed purification protocol, it is important that you estimate fairly accurately how long each step will take. This will necessitate having the correct equipment and reagents available and some degree of foresight. eg. if you plan to use a refrigerated centrifuge but forget to switch it on 30 min prior to use, this will unnecessarily delay the procedure. If you multiply your best guess time estimate by a factor of 2-3, you should be close to a realistic value. Moreover, most published enzyme purification protocols are performed over a period of DAYS (not WEEKS as will occur in LQB681). This may necessitate: your involvement outside the scheduled practical class time of 1-5 pm on Mondays. e.g. transfer of dialysis sacs to fresh medium. adjustment of your protocol to allow for convenient stages at which to halt the separation. i.e. at the end of each prac class, the enzyme MUST be in a solution or precipitate whereby the enzyme activity will not deteriorate appreciably before the following practical class. NOTE: THE EMPHASIS THROUGHOUT THIS UNIT IS ON HANDS ON PARTICIPATION BY ALL MEMBERS OF THE GROUP. If you already are familiar with a particular technique, please allow your practical partners to gain the same experience! Your enzyme purification protocol Your detailed protocol (to be submitted August 11, 2014) should take the form of a FLOW CHART (tree) structure with two columns on the side of the page entitled "buffers required" and "equipment required". On the flow chart, clearly indicate: The DATES where each series of steps will be performed. WHERE reagents will be used, but not concentrations or volumes unless this is crucial to the procedure. WHERE you plan to halt the procedure after each prac class. Ron may modify these! The temperature requirements of steps where temperature control is important to maintain enzyme stability. What you will label each of the samples that you will set aside for evaluation of protein concentration and enzyme activity. Indicate on the flow chart, reminders to record the volumes of each fraction as the purification is performed. The flow chart also should include: A detailed list of all the reagents and equipment, listed on a week-by-week basis. Note that even though you are providing a “draft” list of your requirements here, your group still must supply a copy of materials required EACH WEEK to the prep. Staff before 10am on the FRIDAY PRIOR to that prac class. A detailed account of the assay procedure you will use; the principle, the reaction, temp., substrates, volumes and timing of the assay. A list of relevant references which you used to prepare your flow chart. Your final report The report must be typed, no longer than 12 pages including the BALANCE SHEET, and the chromatographic ELUTION PROFILE, other figures, tables and a bibliography. Each student has the option of submitting a GROUP report, an INDIVIDUAL report or any combination of authors ( e.g. 2 or 3 authors who will then receive the same marks). A group report is preferable but the choice is entirely yours. Centrifugation calculations Rotor Capacity Buckets Beckman JA10 Beckman JA14 Beckman JA17 Beckman JA20.1 (litres) 3.0 1.5 0.7 0.48 6 x 500 mL 6 x 250 mL 14 x 50 mL 32 x 15 mL Max Speed (rpm) 10,000 14,000 17,000 20,000 Radius (mm) min 38 35 56 64 ave 98 86 90 89 g at max rotor speed max 158 137 123 115 min 4,260 7,680 18,100 28,600 ave 11,000 18,900 29,100 40,100 max 17,700 30,100 39,700 51,500 Laboratory Safety Rules General rules NO EATING OR DRINKING IS PERMITTED IN THE LABORATORY. NO PIPETTING BY MOUTH. LAB COATS, SAFETY GLASSES & PROTECTIVE FOOTWEAR must be worn AT ALL TIMES. ALL allergies, medical conditions, minor accidents or injuries must be reported to the laboratory demonstrator. ALL exits must be clear of baggage. The preparative room (laboratory 933) is STRICTLY OUT OF BOUNDS to students. Do not touch equipment on the side benches unless so directed. NO PLASTIC EQUIPMENT IS DISPOSABLE. The exceptions are: capped centrifuge tubes and pipette tips. Equipment in the instrument room may be used ONLY IN THE PRESENCE OF A LECTURER, DEMONSTRATOR OR A MEMBER OF TECHNICAL STAFF. All potentially dangerous chemicals should be handled with gloves IN THE FUME HOOD. Disconnect power supplies whilst assembling/disassembling electrophoresis equipment. Waste disposal All infectious material must be discarded into the yellow laboratory waste container. DO NOT POUR ORGANIC SOLVENTS DOWN THE SINK. Labelled discard containers for miscible and immiscible solvents are provided in the hoods. (If in doubt, mix a drop with water and see if it is miscible). Colour Container Examples of discarded items SHARPS CONTAINER All pipette tips, razor blades, CONTAMINATED glass Pasteur Pipettes. Yellow BROKEN GLASS BIN Broken glassware only. Yellow PATHOLOGY WASTE BAG White DOMESTIC WASTE Yellow ALL BIOLOGICAL/ORGANIC and CONTAMINATED MATERIAL, gloves, plastic centrifuge tubes, Eppendorf tubess, weighing trays, wooden spatulas, etc. Paper and all common, uncontaminated "household" rubbish. Clean up Return all clean items borrowed from the preparative room and special equipment e.g. micro pipettes to the side bench . .