2014
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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
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DO NOT REMOVE RELEVANT TEXTS FROM THE LIBRARY. THEY MUST BE AVAILABLE FOR
USE BY ALL OTHER STUDENTS IN THE CLASS!
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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.
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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!
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DO NOT REMOVE any electrical equipment from the cold room. Condensation will form and
this may short-circuit the device!
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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!
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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.
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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.
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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 4C also may be of advantage if your enzyme is cold-labile.
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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.
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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:
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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:
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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:
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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
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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
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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 .
.