Research Methods in Biochemistry (Spring).doc

RESEARCH METHODSIN

BIOCHEMISTRYC7011

Part 1: SPRING 2010

COURSE ORGANISER: John [email protected]

RESEARCH METHODS IN BIOCHEMISTRY

Table of contents:Page

Timetable 3General information 4Lecture synopsis 6Reading list 7Practicals and problems: general 8Practical 1: Spectrophotometry 9Problems class: Nucleic acids 21Practical 2: Immunology 22Practical 3: Electrophoresis 31Practical 4: Radioactivity 35

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Timetable

This course consists of lectures, practicals and a problems class. Lectures and problem classes are on Tuesdays from 12 to 12.50 p.m. and Fridays from 2 to 4 p.m. (usually with a break) in JMS Lecture Theatre. Practicals are on Mondays at 10 a.m. or 2 p.m. in JMS3B3. You will be divided into two groups. For three of the practicals, group A will work in the morning and group B in the afternoon. The fourth practical requires the whole day, so this will be done on two successive Mondays.Not all the slots are used; please note times of lectures, pre-labs and post-labs carefully. Sessions in bold are assessed! Please make use of the time available during practicals and post-laboratory sessions to ask questions.For the problems class in Nucleic Acids and Gene Expression, a set of problems is included in this handbook. You should attempt these before the first session, where they will be explained. In the second session, you will be given a set of unseen problems to answer on similar topics. These will be assessed as part of the course.This is an 18-credit Level 1 course running in terms 2 and 3 (spring and summer). Please note that this handbook covers the spring term only: a separate handbook will be provided for the second part of the course in the summer term.

FRI 22nd Jan 14.00. Spectrophotometry theory Mike Titheradge15.00 Spectrohotometry pre-lab "

MON 25th Jan 10.00 Spectrophotometry practical

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FRI 29th Jan 14.00 Nucleic acids problem class Trevor BeebeeFRI 29th Jan 15.00 Nucleic acids test “TUES 2nd Feb 12.00 Spectrophotometry post-lab Mike TitheradgeFRI 5th Feb 14.00 Immunology Kathy Triantafilou

15.00 Immunology pre-lab “MON 8th Feb 10.00 Immunology practical “TUES 9th Feb 12.00 NO LECTURE FRI 12th Feb 14.00 Electrophoresis Martha Triantafilou

15.00 Electrophoresis pre-lab “MON 15th Feb 10.00 Electrophoresis practical A “TUES 16th Feb 12.00 Immunology post-lab Kathy TriantafilouFRI 19th Feb 14.00 NO LECTURE

15.00 NO LECTURE MON 22nd Feb 10.00 Electrophoresis practical B Martha Triantafilou TUES 23rd Feb 12.00 NO LECTUREFRI 26th Feb 14.00 Radioactivity Mark Paget

15.00 Radioactivity pre-lab "MON 1st Mar 10.00 Radioactivity practical part

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TUES 2nd Mar 12.00 Electrophoresis post-lab Martha TriantafilouWED 3rd Mar 12.00 Radioactivity practical part 2 Mark PagetTUES 16th Mar 12.00 Radioactivity post-lab "

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Enrolment

All first year students in Biochemistry, Molecular Medicine and Molecular Genetics.The course is run by the Department of Chemistry and Biochemistry.

Course objectives

The aim of this course is to introduce you to the basic principles of the more common experimental methods used in biochemistry. The topics that you will learn about in the spring term are: Nucleic acids analysis Immunological methods Electrophoretic techniques Spectrophotometry Use and Measurement of Radioactive isotopes

You will get practical experience of some techniques and you should aim to understand and visualise what is involved when you encounter terms such as "SDS-PAGE", "ELISA", "radio-immunoassay", etc.

We aim to improve your practical skills by: Extending the range of laboratory techniques with which you are familiar. Developing your ability to analyse data obtained in the laboratory using appropriate IT

packages and computer simulations. Providing you with the opportunity to increase your numeracy and ability to solve

problems in the practical reports.

We aim also to develop the key transferable skills that employers expect graduates to possess. The ability to plan work and meet deadlines.

Successful progress through the course depends on your ability to do this. The ability to communicate fluently orally and in writing.

The work you do in your practicals and post-lab classes will develop this. The ability to work effectively as part of a team.

Teamwork is an essential part of practical work. The ability to abstract information and deliver reports of high standard using appropriate

IT applications.

Learning outcomes:

By the end of the course, a successful student should be able to:1. Demonstrate and understanding of the principles of a range of biochemical techniques2. Show hands-on experience with a wide variety of techniques3. Show a basic competency in numeracy and in handling of biochemical data and calculations, and in writing laboratory reports4. Show an understanding of, and be able to apply, basic statistical methods relevant to biochemistry problems

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Learning Methods

The course involves lectures, practical classes, post-lab classes, a problems class and individual study. Attendance at the scheduled classes is compulsory. Lectures are an efficient way of providing information and define the scope of the course. Practical classes and post-lab classes develop your technical and analytical skills.All of these aspects are essential, and past experience has shown that students who attend less than 90% of classes risk failing. However, just attending classes and doing the set work is not all that is expected of you. Individual study, not necessarily confined strictly to the syllabus, is what university life is all about.

Assessment

The assessment consists of an unseen examination at the end of term 3 (60%) and a continual assessment component using the marks from the practical reports and problems class (40%). The problems test must be submitted at the end of the class. Practical reports should be submitted to the Life Sciences Office, JMS3B10, by 4 p.m. on the Thursday following the practical. Submission within 24 hours of the deadline will be deducted 10% of marks. Later submission, or failure to submit, will normally result in the loss of all of the marks for that piece of work. For any piece of late work where you wish to claim mitigating circumstances an impairment form should be completed and submitted to the Student Advice Team in Chichester II. Please note that computer and/or printer failure which results in the inability to produce written work is not considered an acceptable reason for failure to meet deadlines. Absence from the Exercise or Practical Classes will also incur a loss of all marks for that piece of work unless a mitigaing evidence form is submitted to the Student Advice Team.

Collusion and plagiarism in submitted course work is NOT permitted. All work will be scrutinised for evidence of plagiarism and collusion and any submitted work showing evidence of this will be investigated and penalties imposed. It is acceptable, and indeed encouraged, to work together in practicals to obtain data and discuss how to interpret the data. However the calculations and explanations submitted must be your own work and not done as part of a group effort. The latter is collusion and will be penalised as such.

The issue of plagiarism is a growing academic concern, and one that the University of Sussex takes seriously. Up to date information on the University regulations concerning plagiarism can be found at the following link:

http://www.sussex.ac.uk/academicoffice/plagiarism

Course monitoring

Student feedback will be monitored by a questionnaire at the end of the course. However should you feel the need to bring something to our attention earlier than this, please contact the course organiser by e-mail ([email protected]). The questionnaires are included in our Academic Audit file for this course and any constructive comments or suggestions for improvements are considered carefully for adoption in the following year.

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Lecture Synopsis

Spectrophotometry Mike Titheradge Principles of spectrophotometric methods; ultra violet and visible spectrophotmetric methods; the Beer-Lambert Law; the molar extinction coefficient and its measurement; measurement of the concentrations of solutes using the molar extinction coefficient and standard curves; measurement of the activity of an enzyme.

Immunological methods Kathy TriantafilouNature and production of antibodies (monoclonal and polyclonal); primary, secondary and hyperimmunised responses; tagging of antibodies with fluorochromes, radioisotopes and enzymes; immunodiffusion; use of antibodies in protein purification (immunoprecipitation, affinity chromatography); recognition and purification of cells (immunofluorescence, FACS); use of antibodies for antigen localisation (E.M. and confocal microscopy with tagged antibodies) electrophoresis; ELISA and radio-immunoassays.

Electrophoretic methods MarthaTriantafilouDenaturing; discontinuous buffer system; SDS-PAGE; Western blotting; iso-electric focusing; 2-dimensional gel electrophoresis; application of methods to Mr determination, and

protein sequencing.

Radioactive isotopes Mark PagetRadioactive decay and the nature of radioactive emissions; units of measurement ~ DPM & DPS, Ci & Bq; half-life concept; detection & measurement of radiation ~ scintillation counting; efficiency of counting ~ CPM & DPM; counting error; concept of specific radioactivity & general uses of radioactive tracers; auto-radiography; safety aspects. [Radioimmunoassays under “Immunological Methods”].

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Reading List

We do not know of any single textbook which covers all of the topics in this course at the practical level. However the following are useful for several topics:

Wilson, K & Walker, J. Principles and Techniques of Practical Biochemistry (4th ed) Cambridge University Press.Holme, D.J. & Peck, H. Analytical Biochemistry (3rd ed) Longman

Most general biochemistry textbooks contain much of the material - but it is often scattered. Mathew & Van Holde (Biochemistry) has mostly good coverage in the sections "Tools of Biochemistry". Specific recommendations are Hames & Rickwood (eds.) Gel Electrophoresis of Proteins (IRL Press) and chapter 12 (immuno-blotting) in Harlow & Lane (1988) Antibodies - A Laboratory Manual (Cold Spring Harbor) for electrophoresis.

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RESEARCH METHODS IN BIOCHEMISTRYPRACTICALS

Practicals and problems: General Notes

1. Please read the practical schedules before attending the practicals in order to have the purpose of each experiment and the nature of the measurements to be made, clear in your mind.

2. The practicals should be written up and handed in at the Life Sciences office by 4 p.m. on the Thursday following the practical.

3. Safety regulations require all students to wear lab coats whenever they are working in the laboratory.

4. Please note that some of the chemicals you will use are possible health hazards (indicated in the schedules) and you must note and obey the following precautions when using these chemicals. These may be harmful, irritants and even toxic by inhalation, swallowing and skin contact. They must be dispensed using the automatic pipettes provided. Take the greatest of care when dispensing these chemicals and if any contact is made with them, rinse the affected area(s) with copious amounts of water and inform a demonstrator / lecturer. If in any doubt about the use of these chemicals consult a demonstrator / lecturer before their use.

No solutions whatsoever are to be pipetted by mouth. Please wash your hands after handling any of the hazardous chemicals and when you leave the laboratory.

5. If there appears to be any fault(s) with electrical equipment (loose or worn wiring etc.) do not use that equipment and do not try to repair the fault yourself but report it to the technicians and to the demonstrator / lecturer.

6. Use a small notebook (spiral-bound or fixed page) not loose pages to record your observations, data & results. Please note that Sandwich degree students must show this notebook to the placement tutor (Professor A. Moore) at the end of this course.

7. COSHH Regulations 1988 :

These schedules have been subjected to an informal safety assessment. The document advises students of the risks to health arising from their work and the precautions to be taken.

Date: Signed:

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Practical 1: SPECTROPHOTOMETRY

The concentration of a coloured compound in solution can be estimated by measuring the quantity of light it absorbs at a given wavelength (l). This principle forms the basis of very many quantitative methods in biochemistry. The techniques involved come under the general heading of spectrophotometry and the instrument used is a spectrophotometer.

THEORY

The elementary theory of spectrophotometry given here is concerned with the absorption of monochromatic light (i.e. light of a single wavelength) by a single compound in solution. There are two laws of light absorption:

(1) Lambert's law. This states that for a thin layer the same proportion of the incident light is absorbed, whatever the light intensity. Thus if we consider a thick layer (e.g. the 1cm. cell used in most spectrophotometers) and divide it into a series of thin layers, each successive layer absorbs the same fraction of the incident light falling on that layer. Therefore it follows that as light passes through the cell as a whole, the intensity of the light falls off exponentially:

Io - incident light intensityI - emergent light intensityl - length of the cell

This is expressed mathematically as:

I/Io a e-l

or ln I/ Io a l

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or log10 I/Io a l

log10 Io gives a measure of the amount of light absorbed by the solution and is Iknown as the absorbance, A of the solution (also known as the optical density or extinction) at a given wavelength. A 1cm. light path is used in most measurements, and the symbol Anm (or more pedantically A1cm) is often used to indicate the

absorbance of a solution in a 1 cm. cell, at a wavelength (expressed in nm.)

(2) Beer's law. This states that the absorbance (A) is proportional to the concentration of the absorbing solute.

i.e. log10 Io a concentration (c) I

Beer's law often does not hold for concentrated solutions.

By combining Lambert's law and Beer's law, we have:

log10 Io a c.l I

or log10 Io= E.c.l. or A = E.c.l. I

where E is a constant, known as the molar extinction coefficient. It has units of l/mole.cm when c is in moles/litre and l is in cm. E is the theoretical absorbance of a 1M solution, with a 1 cm. light path.

If we know E for the solute of interest, and are using a 1 cm. cell ( l = 1 cm) the concentration of the solute can readily be calculated from the absorbance value (A). E can either be obtained from the literature or, more usually, is determined by measuring the absorbance of a standard solution of known concentration. In the latter case it is not necessary to calculate E directly, a standard curve is often constructed and the concentration of the unknown solution is read off directly from this standard curve. The absorbance value of any solution can be measured directly, using a spectrophotometer.

Many substances of biochemical interest do not absorb light of visible wavelengths. Some can be estimated spectrophotometrically by reacting them with reagents to give coloured compounds (e.g. amino acids can be estimated after reaction with ninhydrin). In such cases it is often necessary to prepare a standard curve. Some substances (e.g. proteins, nucleic acids) absorb ultraviolet light, and can be estimated using this property.

An extinction value of 1.0 means that the light leaving the solution (I) has an intensity 1/l0th of that of the incident light (Io). As the fraction of light not absorbed becomes

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very low (<1/l0th), it becomes difficult to measure accurately. In practice, extinction values of >1.0 should be avoided, both for this reason and because Beer's law does not hold for concentrated solutions (high extinction values). When a value of log10 Io greater than 1.0 is encountered there are two courses: I

(1) If the coloured solute has not been estimated after reacting with a reagent (i.e. if the solute absorbs light itself), the solution may be diluted accurately to give an extinction < 1.0, and read again.

(2) If the solute of interest is not coloured, but is estimated with a reagent to give a coloured product, the estimation must be repeated using a smaller quantity of the solute, so as to give an extinction value of less than 1.0. It is not valid to dilute the solution in this case (why?).

THE SPECTROPHOTOMETER

There are two models of the Ultraspec II spectrophotometers available in the laboratory: the Model 4050 measures light only in the visible region (325-900nm) and the Model 4051 measures light both in the visible and ultraviolet region (200-900nm).

OPERATING INSTRUCTIONS

1. Switch on the mains electrical supply and the power switch at the rear of the instrument. The machine will now carry out a series of automatic calibration checks for approximately 2 mins. Do not open the cell compartment or attempt to use the machine during this period.

When the display changes from CAL to 360nm the instrument is ready to use, although it is better to allow a further 10 min warm up period for the instrument to stabilise before taking readings.

2. If you are using a Model 4050 the Deuterium lamp will remain ON after the initial calibration period. If you are not intending to use wavelengths in the ultraviolet region, switch OFF the Deuterium lamp using the DEUTERIUM ON/OFF key on the front of the instrument as these lamps are very expensive and have a very limited life span.

3. Select required wavelength using WAVELENGTH +/- keys. The longer the key is depressed the faster the rate of change of wavelength. If the wavelength display flashes this indicates the deuterium lamp should be turned on (Model 4050 only). For wavelengths below 325nm switch on the deuterium lamp using the DEUTERIUM ON/OFF key. It will take approx. 3s for the lamp to strike and the instrument should be left for a further 15 mins for the lamp to stabilise before readings are taken.

4. Press the MODE key to select ABS (Absorbance).

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5. Load the reference cell ("blank" sample) into the cell holder position 1 (BLUE). Load the samples to be measured into positions 2 to 6 as required.

6. Press CELL NUMBER key until cell number 1 shows on the display.

7. Press SET REF key to zero the instrument on the reference cell.

8. Using the CELL NUMBER key, bring your samples into the light path and record the absorbance value from the display.

A flashing display indicates that the absorbance measurement is out of the range of the instrument and the sample must be diluted and the absorbance measured again. Note that the display has a maximum value of 3.000 absorbance units, however for accurate results you should not be taking readings at this high an absorbance (max. 2.00, but most accutate below 1.00).

9. If the wavelength of the instrument is changed, then you must re-zero the instrument on the reference cell using the SET REF key.

NB. These instruments are delicate and very expensive and must be treated with care. Never stand cells on the top of the machine and report any spillages in or on the machine immediately.Never touch the optical surfaces of the cells and always wipe the outside surfaces of the cells to ensure that they are clean. Always remove the cells from the instrument to fill them. After use, the cells should be rinsed in distilled water and never left in the spectrophotometer.

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SPECTROPHOTOMETRY

DETERMINATION OF PLASMA LACTATE DEHYDROGENASE ACTIVITY

IMPORTANT

Before attending the practical, please read the section on the theory and practice of spectrophotometry in the preceding introductory notes.

INTRODUCTIONLactate dehydrogenase activity is present in almost all the tissues of the body and is invariably found only in the cytoplasm of the cell. Enzyme activities are very high in tissues compared with the serum (ie 500 fold) and therefore leakage of the enzyme from even a small amount of tissue damage can increase the activity 10-fold. Elevations of the serum levels of lactate dehydrogenase are associated with a number of different disease states, for example myocardial infarctions, liver disease, anaemia, renal disease and muscular dystrophy. Measurement of the activity in the serum is used clinically for both diagnosis of myocardial infarctions, liver disease etc., together with the monotoring of the progression of the disease following treatment.

Lactate dehydrogenase is a hydrogen transfer enzyme which catalyzes the oxidation of L-lactate to pyruvate using NAD+ as the hydrogen acceptor. The reaction is reversible and the reaction equilibrium strongly favours the reverse reaction, namely the reduction of pyruvate to lactate.

CH3COCOOH + NADH + H+ CH3CHOHCOOH + NAD+

pyruvate lactate

The aim of this practical is to design a method for the measurement of lactate dehydrogenase activity in plasma and to measure the activity in the two samples of plasma provided. In the course of this practical you should become familiar with both the operation of the spectrophotometer and the use of spectrophotometry to determine (a) the absorption spectra of a solute, (b) the molar extinction coefficient of a solute and (c) the activity of a given enzyme. (1) Measurement of the Absorption Spectra of NADH

To determine the activity of lactate dehydrogenase you will need first to determine the wavelength at which to measure the production of NAD+.from NADH. You will therefore need to analyse the absorption spectra of both NADH and NAD+. This can only be done on the Ultraspec 4050, therefore if your instrument is an Ultraspec 4051 carry on with preparing the dilutions in part (2) until another machine is available.

Reagents: NAD+ (0.lmM in 0.05M phosphate buffer, pH 7.4).NADH (0.lmM in .0.05M phosphate buffer, pH 7.4).

Procedure: Put 3mls of NAD+ and NADH into 2 clean cuvettes. Measure the absorbance of the two solutions against a blank cuvette containing 0.05M phosphate buffer, pH 7.4 at 20nm intervals between 240 nm and 400nm in the

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spectrophotometer. (NOTE - (i) You must re-adjust the zero on the spectrophotometer each time the wavelength is changed, (ii) you must use the silica cuvettes provided and not plastic cuvettes as these absorb light in the ultraviolet region..

Plot graphs of the absorbance against wavelength for both NAD+ and NADH, and also a difference spectrum (absorbance NADH minus absorbance NAD+) against wavelength. From the difference spectrum determine the optimum wavelength to measure the activity of the lactate dehydrogenase in later experiments.(Consult a demonstrator before proceeding with the next section).

(2) Determination of the Molar Extinction Coefficient of NADH

The activities of enzymes are normally measured in terms of International Units, i.e. µmoles of product formed/minute/ml of enzyme. To determine the change in concentration of the product in this reaction, you will need to determine the Molar Extinction Coefficient of NADH. This can be determined using the Beer-Lambert Law. Reagents : 0.5 mM NADH in 0.05M phosphate buffer, pH 7.4.

Procedure: Make dilutions of the NADH solution provided with 0.05M phosphate buffer, pH 7.4. to give approximately 5 solutions with equally spaced concentrations in the range between 0.05mM and 0.25mM NADH. Each solution should have a final volume of 3 mls. Read the absorbance of each of these solutions in the spectrophotometer against a blank cuvette containing 0.05 M phosphate buffer, pH 7.4. at the optimum wavelength determined in (1) above.

Plot your results on a graph of absorbance against concentration and calculate the Molar Extinction Coefficient (E) for NADH.

(3) Determination of the concentration of NADH in an unknown sample. Procedure: Solution A is a solution of NADH in 0.05M phosphate buffer, pH 7.4.

Estimate the concentration of NADH in Solution A with the standard curve obtained in (2) above. (If necessary dilute samples of Solution A with 0.05M phosphate buffer, pH 7.4.)

(4) Determination of the Activity of Lactate Dehydrogenase in Plasma

Reagents: 0.05M phosphate buffer, pH 7.4; 10mM NADH; 30mM pyruvate; plasma samples B and C.

Procedure: To a clean cuvette (1), add 2.90ml of 0.05M phosphate buffer and 0.lml of water. Mix the contents of the cuvette and place it in the spectrophotometer. Zero the spectrophotometer against this cuvette. In a second cuvette (2), add 2.74 ml of 0.05M phosphate buffer; 0.06ml of 10mM NADH and 0.lml of the

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plasma sample B. Mix by inversion and leave for 2 minutes. Add 0.1ml of 30mM pyruvate, mix rapidly by inversion and place in the spectrophotometer. Read the absorbance of (2) against (1) at the wavelength determined in Section 1 above, at 1 min intervals for 10 mins. The absorbance should decrease with time. Plot the absorbance against time and determine the rate of the reaction by the change in optical density units/min from the line obtained. lf the rate of change of absorbance is either too slow or too fast to obtain an accurate assessment of the initial rate, repeat the assay with more or less plasma, adjusting the volume of the water added to the blank accordingly. Once you are satisfied that you have achieved the optimal conditions, repeat the procedure using the plasma sample C.

Calculate the activities of the enzyme in the two plasma samples in terms of µmoles of NADH oxidised/min/ml of plasma.

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Name ......................... Date .......... Tutorial Group..........................

Data and Comprehension Sheet - Spectrophotometry

(1) (a) Attach graphs of absorbance spectra(b) The optimal wavelength for measuring the formation of NADH was ..........

(2) (a) Attach graph of absorbance vs. concentration of NADH.(b) Distinguish between absorbance and molar extinction coefficient.

(c) The molar extinction coefficient for NADH was ....................

(3) (a) The absorbance of solution A was ..........(b) The concentration of solution A was ...........

(4) (a) Attach graphs of the rate of change of absorbance with time for samples B and C.

(b) Calculate the activity of the lactate dehydrogenase solution in µmoles of NADH utilised/min/ml of plasma.

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(c) If the normal range of lactate dehydrogenase activity in plasma for a healthy individual is between 0.08 - 0.2 Units/ml of plasma, comment on the possible state of health of the individuals from which the two plasma samples were taken. (Note 1 unit of enzyme activity is 1 µmole of product produced/ min/ ml of plasma).

(d) What factors would you expect to affect the accuracy of your measurements?

(e) What factors would you expect to influence the activity of the enzyme?

(5) (a) A 0.05mM solution of NADH was placed in a lcm spectrophotometer cuvette and a beam of light at 340nm was passed through the cuvette. What proportion of light would emerge from the other side of the cuvette?

(b) A solution of Malachite Green has an optimal density of 0.61 when measured in a lcm pathlength cuvette at 616nm. If the Molar Extinction Coefficient of the dye is 5.3x104 litre/mole.cm at 616nm, calculate the concentration of the solution.

(c) Solution D contains a mixture of ADP and CMP. These compounds both absorb light in the same range of the spectrum and are often found together in

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biological extracts. The molar extinction coefficients of these compounds are: At 260nm: ADP, 15.4x103; CMP, 7.5x103 litres/mole.cm At 280nm: ADP, 2.5x103; CMP, 13.0x103 litres/mole.cm

If the following optical densities were measured in a lcm cuvette: At 260nm 1.45 At 280nm 1.89 Calculate the concentrations of ADP and CMP in Solution D, explaining

the principle involved.

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Problems class: NUCLEIC ACIDS AND GENE EXPRESSION

NB: Consult your notes from the Nucleic Acids and Chromatin Structure lectures in Cellular Biochemistry.

Attempt the problems below BEFORE the first session, where they will be explained. In the second session, you will be given an unseen set of problems on similar topics, to be completed before the end of he session.

(1) (a) The partial composition (in mole-fraction units) of one of the strands of a double-helical DNA molecule is: [T] = 0.35; [C] = 0.25. What can be said about

(i) [A] and [G] of the same strand(ii) [A], [C], [G] and [T] of the complementary strand?

(b) In the DNA of certain bacterial cells, 13% of the nucleotides are adenine. What are the percentages of the other nucleotides?

(2) The molecular weight of an E. coli DNA molecule is very great, about 2.5 x 109. The average molecular weight of a nucleotide pair is 660, and each nucleotide pair contributes 0.34 nm to the length of DNA.

(a) Given the above information, calculate the length of an E. coli DNA molecule. Compare the length of the DNA molecule with the actual cell dimensions. How does it fit in?

(b) Assume that the average protein in E. coli consists of a chain of 400 amino acids. What is the maximum number of proteins that can be encoded by an E. coli DNA molecule?

(3) A protein synthesised in rat liver has 192 amino acid residues. What is the minimum number of DNA base pairs required to code for this protein? It is in fact encoded by a gene of molecular weight 9.5 x 105. Suggest possible explanations for this.

(4) Calculate the average length in nm and the average molecular weight of the genes coding for (a) tRNA and (b) ribonuclease (104 amino acid residues) in a bacterial genome.

(5) Given that fruit fly (Drosophila) somatic cells each contain about 0.3 pg of DNA, calculate:(a) The number of cells present in a larva, 5 of which combined together yielded 1 ml of a pure DNA extract giving an absorbance (in a cell of 1 cm path length) at 260 nm of 0.396.(b) The appoximate size of the Drosophila haploid genome in megabase pairs (MBP).(c) The compaction ratio required to contain fruit fly DNA in a cell nucleus 1.5 m long. Assume:(i) DNA recovery from the larvae was 75%.(ii) The extinction coefficient of a 1 mg/ml solution of DNA at 260 nm = 20. (iii) 1 BP has a molecular weight of 660 and a length of 0.34 nm.(iv) DNA is divided equally among 8 chromosomes in fly somatic cells.

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Practical 2: Immunology

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

BACKGROUND

Enzyme linked immunosorbent assay (ELISA) is a solid phase immunoassay in which one of the interacting components, i.e. either antigen or antibody, is fixed to a solid matrix. ELISAs can be designed in an number of formats as quantitative assays for either antibody or antigen, e.g. indirect/sandwich, competitive (see Figs. 1, 2). The most popular form of ELISA is carried out in 96 well micro-titre plates in which the polystyrene acts as an absorbing surface for either antibody or antigen. Proteins (antibody and many antigens) have the property of binding to polymeric matrices such as synthetic plastics or naturally based products like nitrocellulose (used in Western/electroblotting). ELISAs are technically advantageous since all the reactions (antibody-antibody binding, detection of antibody-antigen complexes) can be carried out in the wells of the plate. In addition, microtitre plates can be manufactured with degrees of precision and reproducibility such that all the wells in a plate can act as identical minicuvettes for spectrophotometry. Dedicated instrumentation has been developed, e.g. ELISA plate readers (modified spectrophotometers), ELISA plate washers. As a result, ELISAs have become cheap, sensitive and convenient assays of biological materials particularly in routine clinical laboratories.

An indirect ELISA is an example of a ligand binding assay, the resulting plot of antibody titre (usually a function of dilution of the original serum) against absorbance readings having the shape seen in Fig. 3 (N.B. the use of semi-log paper to cope with the wide range of values of serum dilutions).

ELISA FOR ANTIBODIES TO BOVINE SERUM ALBUMIN.

As a demonstration of the ELISA technique we will use an indirect format to detect antibodies to serum albumin (BSA). In simple terms, plates will be "coated" with BSA, exposed to a range of dilutions of anti-BSA antibodies (raised in the rabbit) (first antibody) to produce a binding or titration curve which can then be used as a calibration or standard with which to determine the "titre" of anti-BSA antibodies in other rabbit sera. The presence of rabbit IgG antibody bound to BSA in the wells will be detected by antibodies to rabbit IgG (raised in the goat) conjugated to the enzyme horse radish peroxidase (second antibody conjugate) using o-phenylenediamine (OPD), a substrate which the peroxidase converts to a coloured soluble product.

An important step in the ELISA is to block sites in the wells to which BSA has not bound, otherwise considerable proportions of the subsequent additions of primary antibody and second antibody conjugate will bind non-specifically and mask the specific antibody-antigen reactions we wish to produce and detect quantitatively. This can be achieved in a number of ways (e.g. with a non-reacting protein), but we will use a simple and convenient procedure with phosphate- buffered-saline containing the nonionic detergent Tween 20 (PBS-Tween).

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Not only does the Tween bind to sites which have absorbed no BSA, but the PBS-Tween acts as solution for washing wells and diluting primary and second antibodies.

ELISA plates contain 96 wells in an 8 x 12 format. Each well has an optically flat bottom to allow the vertical passage of light in a dedicated spectrophotometer (ELISA Reader). However, the convention is not to use the outer wells since these may be at a different temperature from those in the interior of the plate (Edge Effect). Hence, in practice a 10 x 6 format is normally used. The design of and procedure for the ELISA for anti-BSA antibodies is summarised in the diagrams below. The four main steps have been previously optimised for maximum sensitivity in terms of concentration of BSA for coating and the dilution range for the first antibody and the second antibody-conjugate.

The four steps are as follows.

A. Coating -Plate

Solutions: coating buffer - 0.1 M bicarbonate buffer, pH 9.610 g/ml solutions of bovine serum albumin(BSA), chick ovalbumin (OA) and bovine lactalbumin (LA)

Pipette 100l of coating buffer, BSA, OA and LA solutions into wells marked Blank, BSA, OA and LA respectively

2 3 4 5 6 7 8 9 10 11B Blank BSA BSA BSA BSA BSA OA OA LA LAC Blank BSA BSA BSA BSA BSA OA OA LA LAD Blank BSA BSA BSA BSA BSA OA OA LA LAE Blank BSA BSA BSA BSA BSA OA OA LA LAF Blank BSA BSA BSA BSA BSA OA OA LA LAG Blank BSA BSA BSA BSA BSA OA OA LA LA

Write your name on side of plate.

Wrap plate in a sheet of plastic film and incubate at 4o C (in fridge) overnight.

Next day empty contents of wells by flicking the plate several times over sink. Wash wells by filling them with PBS-Tween from a wash-bottle and then empty by flicking over sink. Repeat this washing procedure twice. After the last wash remove residual liquid from the wells by repeated "banging" (!) of the inverted plate onto a pad of tissue paper placed on the bench. Ensure that the wells are free from bubbles at the end of this treatment.

B. Addition of first antibody, unknowns and controls/blank

Solutions: PBS-Tween1 in 5 dilution of rabbit anti-BSA serum in PBS-Tween

24


(i.e. 1 volume serum + 4 volumes PBS-Tween)"Unknown" dilutions of rabbit anti-BSA serum (U1, U2)

First, prepare doubling dilutions of the anti-BSA serum (first antibody) according to the instructions below

Dl(1:5) Designate the 1:5 dilution of anti-BSA as Dilution 1(D1). Prepare doubling dilutions from Dl as follows.

D2(1:10) Pipette 1 ml of Dl into a tube containing 1 ml of PBS-Tween. Mix thoroughly.

D3(1:20) Pipette 1 ml of D2 into a tube containing 1 ml of PBS-Tween. Mix thoroughly.





DS(1:640) Pipette 1 ml of D7 into a tube containing 1 ml of PBS-Tween. Mix thoroughly.




D12(1:10240) Pipette 1 ml of D 11 into a tube containing 1 ml of PBS-Tween. Mix thoroughly.

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Add 100 l of these solutions to wells as shown in the diagram below.

2 3 4 5 6 7 8 9 10 11B PBS.

TD12 D12 D6 D6 U1 D1 D1 D1 D1

C PBS.T

D11 D11 D5 D5 U1 D2 D2 D2 D2

D PBS.T

D10 D10 D4 D4 U2 D5 D5 D5 D5

E PBS.T

D9 D9 D3 D3 U2 D8 D8 D8 D8

F PBS.T

D8 D8 D2 D2 PBS.T

PBS.T

PBS.T

PBS.T

PBS.T

G PBS.T

D7 D7 D1 D1 PBS.T

PBS.T

PBS.T

PBS.T

PBS.T

Wrap the plate in plastic film and leave undisturbed on the bench for 1 hour.

This arrangement provides the following:

a. Enzyme-substrate blank (no antigen/antibodies in wells) - wells 2B-G (could be used if necessary to blank ELISA Reader).

b. Duplicates of the 12 dilutions of the rabbit anti-BSA serum (wells 3,4B-5,6G) with which to form calibration/binding curve.

c. Unknown dilutions Ul and U2 in duplicate (7B,C; 7D,E).d. Test of antibody specificity - OA and LA in wells exposed to 4 dilutions of

rabbit anti-BSA serum (wells 8,9 B-E; 10,11 B-E).e. Test for non-specific binding of second antibody conjugate to antigens

(wells 7 F-G; 8,9 F-G; 10,11 F-G).

After 1 hour flick contents of wells into sink and wash three times with PBS-Tween as before. N.B. remember to "bang" inverted plate onto pad of tissues to remove residual liquid from wells.

C. Addition of anti-rabbit IgG-peroxidase conjugate (and PBS-Tween control).

Solutions: PBS-Tween1:15000 dilution of goat anti-rabbit IgG-peroxidase conjugate (second antibody-conjugate)

Using an 8-channel pipette -

First. dispense 100 l of PBS-Tween into wells in row 2 (including edge wells - for speed).

Second, using the 8-channel pipette add 100 l of second antibody-conjugate to wells in rows 3-11 (including edge wells).

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Now, wrap the plate in plastic film and leave undisturbed on the bench for 1 hour.

After 1 hour flick contents of wells into sink and wash three times with PBS-Tween as before. N.B. remember to "bang" inverted plate onto pad of tissues to remove residual liquid from wells.

D. Addition of enzyme-substrate solution.

Solution: citrate/phosphate buffer, pH 5, containing 40 mg OPD/100 ml

Immediately prior to use add 4 l H2O2 to 10 ml of citrate/phosphate-OPD solution. CARE - WEAR GLOVES - H2O2 burns and OPD is toxic. Using an 8-channel pipette add 100 l of the OPD/H2O2 solution to the wells in rows 2-11 (including edge wells).

Cover the plate with a sheet of tin foil and leave undisturbed on the bench for 30 minutes.

Then stop enzyme reaction by adding 50 l of 2M H2S04 to wells in rows 2-11 using an 8 channel pipette.

The absorbance at 492 nm in wells 2B-G to 11B-G is read using the Anthos ELISA Plate Reader.

For TASKS see over

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28


RESEARCH METHODS IN BIOCHEMISTRYPractical 2: Immunology

Name ...................................... Tutorial Group .............................

From the ELISA Reader Print-Out calculate the average of the duplicate readings.

(1) Controls Note values of controls.

a. Enzyme-substrate control, wells 2B-G. From the readings given by these wells what do you conclude about the suitability of the enzyme substrate solution for this immunoassay?

b. Wells containing BSA, OA, LA but exposed only to second antibody conjugate, i.e. 7FG; 8,9 FG; 10,11 FG respectively. From the readings given by these wells what do you conclude about the degree of cross reactivity by the second antibody-enzyme conjugate ?

(2) Calibration/binding curve.Using semi-log paper, plot Absorbance (linear scale) against anti-BSA serum dilution (log scale); for latter use reciprocal of serum dilution, e.g. 1/5 = 0.2 (as in Fig. 3).

(3) Specificity of anti-BSA serum.On same graph as (2), plot Absorbance readings for reaction of anti-BSA serum against OA (wells 8,9 B-E) and LA (wells 10,11 B-E).What do you conclude about the specificity of the anti-BSA serum ?

(4) Determination of anti-BSA serum in dilutions Ul and U2.Using the calibration/binding curve calculate the dilutions of anti-BSA serum in:

Ul = dilution of 1/U2 = dilution of 1/

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Attach your calibration/binding curve plots and ELISA Print-Out.

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31


Practical 3: Electrophoresis

SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS

METHOD FOR MINI-GELS

1. You are provided with an apparatus with two sets of plates and two preformed separating gels. An overlay of butanol with bromophenol blue has been placed above each polymerised gel.

There is ONE gel is for each group.

2. Preparation of Stacking Gel

(i) Make up STACKING GEL solution by adding the following to a small Buchner flask:

WEAR GLOVES – ACRYLAMIDE IS A NEUROTOXIN

1 ml. STACKING GEL acrylamide solution (20 g monomer + 1 g bis/ 100ml.)

0.625 ml. STACKING GEL buffer (1 M Tris/HCl, pH 6.83) 2.8 ml. distilled water 0.5 ml. glycerol (use lml. tip with point cut off) 50 l 10% SDS 2.5 l phenol red solution

(ii) Mix and then add 50 l APS (freshly made ammonium persulphate, 10g in 100ml of water) and 10 l TEMED. Mix again.

(iii) Remove butanol overlay from top of separating gel using syringe with green needle and wash top of separating gel by pouring approx. 0 5 ml. of stacking gel solution into top of tray, rock and remove solution with syringe.

(iv) Now fill remainder of gel tray with stacking gel solution. (v) Insert white comb so that the top notches on either side of the comb rest on top

of the gel plates. If necessary, add more stacking gel solution.(v) Allow to set for 15-30 min.(vi) Prepare samples.

3. Running Gels

a. (i) Remove comb from stacking gel by carefully pushing it upwards out of top of tray - wells should be visible.

(ii) Invert assembly and remove excess liquid from wells (and any in upper electrode reservoir) by gentle shaking.

(iii) Set assembly upright again and refill wells with electrode (running) buffer.

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(iv) Pour electrode (running) buffer (29 g glycine + 6 g Tris + 1 g SDS/ 1000 ml.; pH 8.3) into the upper electrode reservoir until the level reaches the top of the glass plate.

b. Proteins supplied have been dissolved (0.25-0.5 mg/ ml.) in SDS-sample buffer (1 ml. 10% SDS, 1 ml. glycerol, 83 l. 1 M Tris/HCl, pH 6.8, 77 mg dithiothreitol to 5 ml with water) and placed in a boiling water bath for 3 min. Bromophenol blue was then added as a tracker dye.

c. Use a microsyringe to underlay at the bottom of the appropiate wells the volumes indicated below. Wash out syringe with distilled water between protein samples.

l . M.Wt.Well 1 albumin - 5 66000 2 ovalbumin - 5 45000 3 glyceraldehyde-

3-phosphate dehydrogenase 5 36000 4 carbonic anhydrase - 5 29000 5 trypsinogen - 5 24000 6 trypsin inhibitor - 5 20100 7 lactalbumin - 5 14200 8 mixture of all 7 proteins - 10 9 H1 histone - 5 see below 10 immunoglobulin G - 5 see below

d. After each group has loaded their samples, carefully transfer the gels to the electrophoresis tank.

e. Add electrophoresis buffer to half-fill the tank.

f. Place cover carrying the electrode connections onto gel apparatus and transfer to electrophoresis safety box. Attach power cables from the box to the power pack, switch on and adjust to 200 volts.

(LUNCH BREAK?)

g. When the boundary of the red dye (with the bromophenol blue marker dye from each well just behind it) reaches the bottom of the gel (in about 30 min.), switch off the power pack, open safety box and remove the covers from gel apparatus. Remove clamps and detach gel trays from gaskets - N.B. GEL ASSEMBLY WILL BE HOT ! Pour off buffer from upper electrode reservoir.

4. Staining Gels

a. To remove gel, first place tray on bench with glass plate uppermost. Insert a spatula between glass and alumina plates and twist it until the plates separate leaving the gel on the alumina plate (not the easiest of manipulations but it does work in the end !). Squirt water from a wash bottle between gel and glass plate and carefully lift gel off and transfer it to a staining dish. Mark your dish.

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b. Stain for 15 min. in 0.25 %(w/v) Coomassie Brilliant Blue in methanol/water/acetic acid (45:45:10 v/v/v) using rocking platform. Pour off dye solution and destain using approximately 5 changes of destaining solution (methanol/acetic acid/water: 30/10/60: v/v/v), allowing 10 minutes between each change of solution, or until bands become visible and background stain is negligible. Do not over destain as the intensity of the protein bands willalso decrease.

c. Place stained gel on glass plate and with a ruler measure distance moved down the gel by the major bands from each well; also measure the distance travelled by the bromophenol blue tracker dye and use this value to calculate the relative mobility for each band (as the ratio - band distance/dye distance).

TASKS.

(1) Draw a table with the distance (mm) migrated for each band, the distance migrated by the dye front (mm) and the relative mobility for each band.

(2) Plot relative mobilities of protein bands in well 8 on log/linear paper to form a calibration line and compare mobilities of individual proteins in wells 1-7.

(3) The sample in well 9 was obtained commercially as an apparently purified preparation of H1 histone (M.Wt. from primary structure = 22000). Comment on the result you obtain (M.Wt. of other histone types - H2b, H3, H2A, H4 = 14000, 14000, 12500, 11000 respectively).

(4) The sample in well 10 consists of sheep immunoglobulin G (IgG). How many protein components do you observe after SDS-electrophoresis and calculate their approx. M.Wts.? What does the SDS-electrophoresis pattern tell you about the composition of this particular protein?

REFERENCES:

Hames & Rickwood (1981) "Gel Electrophoresis of Proteins", IRL Press, Oxford.Harlow & Lane (1988) "Antibodies - A Laboratory Manual" Spring Harbor - Chp. 12 (Immunoblotting).

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35


PRACTICAL 5: RADIOACTIVITY

USE OF RADIOACTIVE ISOTOPES

SAFETY

HUMAN BLOOD AND RADIOACTIVE ISOTOPES are used in this experiment.

Be especially careful throughout the practical and note that:

(a) you must wear gloves whilst handling these materials;

(b) ALL waste material & used vessels must be placed only in the designated receptacles;

(c) every vessel must be clearly labelled;

(d) any excess radioactive material or blood must be returned to the demonstrators;

(e) any accident, however trivial, must be reported to the demonstrators immediately.

IF YOU ARE UNSURE ABOUT ANY ASPECT OF THE PRACTICAL,

ASK A DEMONSTRATOR FOR HELP

This experiment provides an example of the use of radioactive isotopes, showing how easily they can give quantitative measurements of parameters that would be difficult, or impossible, to evaluate by other means. It is particularly important that you become familiar with the form in which the data are obtained and the calculations required to make use of them.

The aim of the experiment is to measure the uptake of the amino acid L-serine into erythrocytes, in terms of intracellular concentration per given time. This will involve dual labelling, with L-[3-3H]serine used to monitor the amino acid flux and [14C]sucrose to measure residual extracellular volume of cell pellets. Note: sucrose is NOT taken up into the cells. Total volumes of the pellets will be measured as the difference between wet and dry weights, thus enabling intracellular volumes to be calculated. The effects of some cations and other amino acids on serine influx will be examined.

PROCEDURE

1. You should have a water bath that is set at 37o C – do not alter this.

2. Add suitable amounts of ice and water to the plastic tray for the Eppendorf tube rack.

3. Keep your bottle of isotonic KCl in ice.

4. Label 27 SCINTILLATION COUNTING VIALS (near the tops): (1s, 2s...12s) (1,2..12) and (A1, A2, B).

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5. Label 12 EPPENDORF TUBES on the lids (1-12) and then record their weights to the nearest 0.0001g (see "RESULTS" page). The tubes must be dry!

6. Carefully add the indicated solutions to each Eppendorf tube according to the protocol: follow the order given from top to bottom.

(The L-serine* solution is RADIOACTIVE ~ obtain it from a demonstrator.)

ProtocolEppendorf tube 1 2 3 4 5 6 7 8 9 10 11 12Addition µl µl µl µl µl µl µl µl µl µl µl µl

Water 400 400 200 200 200 200 200 200 200 200 400 400Medium 2Na 500 500 500 500 500 500Medium 2K 500 500 500 500 500Medium 2Li 500Glycine (10 mM) 200 200L-alanine (10 mM) 200 200D-alanine (10 mM) 200 200b-alanine (10 mM) 200 200

L-serine* (1 mM) 100 100 100 100 100 100 100 100 100 100 100 100

2Na -medium: 290 mM NaCl, 4 mM MgSO4, 40 mM Tris / HCl (pH 7.6 at lab. temp.)2K- and 2Li-media are the same except that NaCl is replaced with KCl or LiCl, respectively.

L-serine* contains L-[3-3H]serine. Total concentration = 1mM.Final wash solution is 155 mM-KCl plus about 1 mM sucrose and some [14C]sucrose.

7. Cap the tubes carefully, MIX THE CONTENTS of each well and then place them in the rack in the ice-water tray.

8. Add 20 µl of the L-serine* solution to each vial A1 and A2. (Then RETURN any SURPLUS solution to a demonstrator.)

9. Add 100 µl of the erythrocyte suspension (obtain from demonstrator) to each

Eppendorf tube (1 - 12); cap each tube and MIX ITS CONTENTS. (Avoid breaking off the caps when opening and closing the cold tubes.) PROCEED TO THE NEXT STEP QUICKLY.

10. At time zero (start timing) transfer the rack of tubes to the 37o C water-bath.

11. After 20 minutes, return the rack of tubes to the ice-water bath to cool for 3 minutes.

12. Then transfer the tubes to the microfuge and centrifuge them (top speed) for about 20 seconds.

13. Remove the supernatant solutions by aspiration, being careful to avoid losing cells.†

(P ractise this technique before starting the experiment.)

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14. Add 1 ml of the cold isotonic KCl solution to each tube, close the caps, mix the contents of each vigorously until all the cells are resuspended, and repeat steps 12 & 13. This is an important step to remove any L-serine that has not been taken up into cells,

15. Repeat step 14. (Second wash.)

16. Add 1 ml of the Final Wash Solution (radioactive ~ obtain from a demonstrator) to each tube; cap the tubes, resuspend the cells, and centrifuge the tubes for about 1 minute.

17. Transfer 400 µl of each supernatant solution to the corresponding counting vial (1s - 12s).

18. CAREFULLY remove as much as possible of the remaining supernatant solutions by aspiration, taking care to avoid losing cells.† Cap the tubes, make sure their outsides are dry and then RECORD their weights (see "RESULTS" page).

19. Add 300 µl of 0.1 % Triton X-100 to each cell pellet and mix the cells well with the solution. Then add 300 µl of 10 % TCA solution (CARE!) to each tube, mix their contents thoroughly and centrifuge them for 1 minute.

20. Transfer 400 µl of each clear supernatant solution to the corresponding counting vials (1 - 12).

21. Carefully remove most of the remaining supernatant solutions and leave the tubes (OPEN) in the metal rack. Ensure that your rack is clearly marked with your Bench label. These tubes will be incubated in an oven to dryness, allowing you to obtain dry weight.

22. Add about 3.6 ml of scintillation cocktail to each vial, including A1, A2 and B, from the "automatic" dispenser. Cap the vials (check that the caps are fully inserted), mix their contents THOROUGLY, and place them in order 1-12, 1s-12s, A1, A2, B, in the counting racks provided. (Note that these racks are NOT symmetrical.) Ensure that your racks are marked with your Bench label and then give them to a demonstrator.

23. PUT YOUR GLOVES IN THE BAGS PROVIDED AND WASH YOUR HANDS BEFORE LEAVING THE LABORATORY.

Your Eppendorf tubes will be dried over-night at 105o C and your vials will be "counted" over the next two days.

At the time noted in the pre-lab talk, return to the laboratory

(a) to collect your DPM results and

(b) to weigh your dried tubes (see "RESULTS" page).

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39


RESULTS

Record your results in the table on the "RESULTS" page. Please print out the results page fill it in and hand it in with your report. Include in your report BOTH the raw data (first results page) and the final calculation sheet.

REPORT

(a) Calculate the following values (see appendix) and record them in the table on the "RESULTS" page. [50 marks]

Specific activity of serine (S.A.) [DPM / nmol]

Residual extracellular volume per tube (ECV) [µl]

Intracellular volume per tube (ICV) [µl]

Cell dry weight per tube (Dry wt.) [mg]

ICV per dry weight of cells [µl / mg]

Serine uptake in 15 min. [nmol / ml ICV]

IMPORTANT NOTE: an excel spreadsheet will be supplied in order to allow you to rapidly analyse your results. However, you should show your calculations for ONE sample – Eppendorf Tube 1 in the final units of nmol / ml ICV.

(b) Plot your values of serine uptake as a bar diagram. [20 marks]

Note: you can draw this by hand or use excel. Remember to include a legend that

explains what each bar refers to as well as labelled axes and a title.

(c) Explain what you conclude from YOUR results, taking into consideration:

(i) the variation between your duplicate measurements (tubes 1 & 11, experimental

error);

(ii) the effects of external cations;

(iii) the effects of other amino acids;

(iv) the highest intracellular concentration of L-serine recorded relative to the final

concentration in the incubation medium.[30 marks]

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APPENDIX

IMPORTANT NOTE: an excel spreadsheet will be supplied in order to allow you to rapidly analyse your results. However, you should show your working out for ONE sample – Eppendorf Tube 1 in the final units of nmol / ml ICV.

(a) Record all DPM values to the nearest WHOLE NUMBER (i.e. no decimal places) and be careful to distinguish values for 3H (serine) from those for 14C (sucrose).

(b) S.A. of serine = [0.5 (A1 + A2) - BH] DPM per 20 µl of a 1 mM solution

= [0.5 (A1 + A2) - BH] / 20 DPM / nmol serine

Explanation – you have a known concentration of serine and you have measured the dpm for a known volume (or known amount in nmol). This allows you to work out the S.A. How much serine is in 20 l of a 1 mM solution? 1 mM means 1 mmol per litre. Working backwards this is equivalent to 1 mol per ml or 1 nmol per l. You added 20 l and therefore 20 nmol serine to each counting tube. Therefore to calculate the specific activity you first work out the mean dpm for 20 nmol, then divide by 20 to give you dpm/nmol serine.

(c) Cell dry wt. = 1000 [ W3 - W1] mg / tube

Explanation – subtract the weight of the tube at the start of the experiment from the total weight of the tube plus dried pellet and you are left with the dried pellet in g. Multiply by 1000 to give you the weight in mg.

(d) ECV + ICV = 1000 [ W2 - W3] µl / tube (1 µl water weighs 1 mg)

Explanation – this is the total volume of liquid in the pellet after the final wash. Some liquid surrounds the cells and there is the aqueous intracellular volume to consider as well. By subtracting the dry weight from the total wet weight, you are left with the total aqueous weight. If we consider 1 µl water weighs 1 mg then you can convert this weight in mg to l.

(e) Total cell extract = [ 0.6 + W2 - W3] ml / tube

Explanation - after the final wash you resuspended the pellet in 600 l. (300 l Triton X100 and 300 l TCA). The total aqueous volume is therefore 0.6 ml plus [ECV+ICV]. Remember to keep the units the same – in this case we are using ml so the ECV + ICV is also in ml.

(f) For sucrose

Supernatant has [ SC - BC] DPM per 0.4 ml or {[ SC - BC] / 0.4 } DPM / ml

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Cell extract has {[ EC - BC][ 0.6 + W2 - W3] / 0.4 } DPM / tube

Hence ECV = 1000 [ EC - BC][ 0.6 + W2 - W3] µl / tube

[ SC - BC]

Explanation – its simple to work out the total ECV + ICV as shown above – but what

proportion of this volume is intracellular and what proportion is extracellular? This

calculation helps you work this out. You sampled 0.4 ml of the final wash after pelleting the

cells (Sc). To work out the dpm in this sample you need to subtract the background count to

give you dpm per 0.4 ml. Then work out the dpm would for 1ml by dividing by 0.4 (same as

multiplying by 1/0.4). For the cell extract, again subtract the background, then you have

your dpm for 0.4 ml (the volume you added to the counting vial) of the total cell extract

([ 0.6 + W2 - W3] ml / tube – see above). Therefore the calculation {[EC - BC][ 0.6 +

W2 - W3] / 0.4 } will give you dpm per tube.

(g) and ICV = {1000 [ W2 - W3] - ECV} µl / tube

(h) For serine

cell extract has [ EH - BH] [0.6 + W2 - W3] / 0.4 DPM / tube

Hence serine uptake is : 1000 ( EH - BH)(0.6 + W2 - W3) nmol / ml ICV

0.4 (S.A.) (ICV)

Be careful with the units - they have been chosen to give convenient values for the results;

but this entails switching from ml to µl and g to mg, etc.

Calculate the values of (ICV / dry wt.) to 1 decimal place only. This ratio is expected to be

approximately constant ( » 2 / 1); if it is not, the most likely cause is a calculation error, so

CHECK YOUR DATA & CALCULATIONS IF IT VARIES MUCH. (Confusion between

DPM from 3H and 14C is another common source of error.)

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Name Bench label Date

Results

WEIGHT OF TUBE (g) RADIOACTIVITY IN VIAL (DPM)

Tube orVial

Empty (W1)

Plus Cells(W2)

Dried( W3)

SupernatantSC [

14C]ExtractEC [

14C]ExtractEH [

3H]1

2

3

4

5

6

7

8

9

10

11

12DPM [14C ] DPM [3H]

A1A2B

Calculated values

Specific radioactivity of serine* was ................................DPM / nmol

Tube ECV µl

ICVµl

Dry weightmg

ICV / Dry wt.µl / mg

Serine uptakenmol / ml ICV

1

2

3

4

5

6

7

8

9

10

11

12

Research Methods in Biochemistry (Spring).doc

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