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_______________________________________________
MOAC Doctoral Training Centre
November/December 200 8
CH921:
Data Acquisition I
Biophysical Techniques
Course Information
Course leader: Dr. Ann Dixon
Contributors: Prof. Alison Rodger; Dr. Claudia Blindauer;
Dr. Steven Brown; Dr. Vilmos Fulop, Dr. Sue Slade
CH921: Biophysical Techniques
Table of Contents Nov/Dec 2008
TABLE OF CONTENTS.
Deadlines and other important information
Timetable
Essay assignment
Laboratory and workshop manual:
Aims and Assessment
Basic Laboratory Skills
Introduction to Protein Databases Workshop
Dichroweb CD Data Fitting Workshop
Experiment I
Experiment II
Experiment III
Experiment IV
Experiment V
DNA Melting Curve Exercise
2
Page
3
4
5
6
7-8
9-10
11-12
13-15
16-17
18
19-23
24
25
26-27
CH921: Biophysical Techniques
Deadlines Nov/Dec 2008
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 Mrs. Monica Lucena 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.
 IAMBEC students to submit all work to Mrs. Christina Forbes.
 MOAC students to submit all work to Mrs. Monica Lucena.
 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. 1% / hour late; 3%/day late unless written extension from Dr. A. Dixon.
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Workshop problems/proof of completion:
Laboratory reports:
Essay:
DNA melting exercise:
NMR assessment:
Due by 5 pm on the day of the workshop.
Due Monday 8th December by 12 noon.
Due Tuesday 25th November at 12 noon.
Due Tuesday 25th November at 12 noon.
Due Tuesday 2nd December at 12 noon.
An oral examination (with 2 hours minutes written/reading work before hand, to be submitted at
the oral) will take place on Monday 1st 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%
3
CH921: Biophysical Techniques
Timetable Nov/Dec 2008
CH921 TIMETABLE. Timetable includes times, dates, academic lecturer, and location
Week 6:
Mon. Nov. 3
Tues. Nov. 4
Week 7:
Mon. Nov. 10
Tues. Nov. 11
Week 8:
Mon. Nov. 17
Tues. Nov. 18
Week 9:
Mon. Nov. 24
Tues. Nov. 25
Week 10:
Mon. Dec. 1
Tues. Dec. 2
Week 10+1:
Mon. Dec. 8
9:00
10:00
UV workshop
Rodger MOAC
Mass Spectrometry
Slade MOAC
11:00
12:00
Fluorescence lecture
& workshop
(DNA melting curve)
Rodger MOAC
Pre-lab
MOAC
(12:30)
11:00
12:00
13:00
14:00
15:00
CD lecture
Rodger MOAC
16:00
Dichroweb
workshop
Rodger
Laboratory: Experiments I & II
Dixon Chemistry B309
9:00
10:00
13:00
14:00
15:00
16:00
Linear Dichroism
Databases workshop
Rodger MOAC
Dixon MOAC
Pre-lab
Laboratory: Exp. III
MOAC
Dixon Chemistry B309
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
Introduction to NMR
NMR Group Work
Brown MOAC
Brown MOAC
Pre-lab
Laboratory: Exp. IV
MOAC
Dixon Chem B309
Solid State NMR Tutorial (Brown): Group 1: 10:00-11:00 am, Group 2: 11:15-12:15 am
Solution State NMR Tutorial (Prokes):
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
Bio-applications of high field NMR
X-Ray crystallography
Blindauer MOAC
Fulop MOAC
Deadline: Essay and DNA melting
12:30
Laboratory: Exp. V
exercise due (12:00)
Pre-lab
Dixon Chem B309
MOAC
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
EXAM
Deadline: NMR Assessment due
(12:00)
9:00
10:00
11:00
Deadline: Lab reports &
FEEDBACK FORM due (12:00)
12:00
4
Crystallography demo/practical
Fulop Biol. Sci.
13:00
14:00
15:00
16:00
CH921: Biophysical Techniques
Essay Nov/Dec 2008
ESSAY. Discuss the discovery of the structure and function of monoclonal antibodies with
reference to a particular protein of your choice. Write your essay from the viewpoint of the
biophysical techniques used to characterize them. Include in your essay (this means it must be a
structured piece of text that hangs together not isolated notes on different techniques – it must tell a
‘story’) consideration of how NMR, absorbance spectroscopies, X-ray crystallography and mass
spectrometry were/might have been used.
Write using an American Chemical Society Journal template (such as Biochemistry, see below for
appropriate web sites).
Use at least 3 primary literature references.
Write 15002000 words plus any diagrams (Figure captions do not count in word count). Make sure
all tables and figures are self contained and also make sure all tables and figures are referred to in
the text.
Marks will be given for content and also spelling, grammar, setting out etc.
Both an electronic and hard copy version should be submitted.
Web sites to lead you to the Biochemistry template and instructions
http://pubs.acs.org/
http://pubs.acs.org/about.html
http://pubs.acs.org/journals/bichaw/index.html
https://paragon.acs.org/paragon/application?pageid=content&parentid=authorchecklist&mid=ag_bi.
html&headername=Author%20Guidelines%20-%20Biochemistry
https://paragon.acs.org/paragon/application?pageid=content&parentid=authorchecklist&mid=mt_bi.
html&headername=Biochemistry%20Templates
All references you use should be relatively recent.
5
CH921: Biophysical Techniques
Laboratory and Workshop Manual Nov/Dec 2008
____________________________________
MOAC Doctoral Training Centre
CH921
Data Acquisition I
Biophysical Techniques
Laboratory and Workshop
Manual
6
CH921: Biophysical Techniques
Introduction and Assessment Nov/Dec 2008
INTRODUCTION. The aims of this lab course are to familiarize you with biological samples and
teach you competence in a set of standard techniques. These include:

COSHH and solution preparation (Basic Lab Skills and Experiment I).

Protein and DNA sample preparation (Experiment II).

Protein concentration determination (Experiment III).

Protein secondary structure determination by circular dichroism (Experiment IV).

Equilibrium binding constant determination by fluorescence spectroscopy: DNA and
ruthenium tris(1,10-phenanthroline) (Experiment V).
* 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.
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:
o Improvement of laboratory skills with respect to sample handling
o Tidiness and cleanliness
o Accuracy of results
o Care of equipment
o Organisation 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.
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CH921: Biophysical Techniques
Aims and Assessment Nov/Dec 2008
FORMAT FOR LAB WRITEUPS. Include the following sections in your reports:
I. Background/Introduction
∙ Purpose of the experiment.
∙ Important background and/or theory.
∙ Description of specialised equipment.
∙ Justification of experiment's importance.
II. Methods
∙ Briefly describe procedure and any modifications to procedure (don't rewrite lab script,
nor say 'see laboratory script').
III. Experimental Results
∙ Present data in tables and graphs (don't forget to label all axes, number figures, and
provide titles).
∙ Use sentences to draw attention to key points in tables or graphs.
∙ Provide sample calculations.
∙ State key result in sentence form.
IV. Discussion
∙ This is the most important part of the report, where you can show your understanding of
the experiment. 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?
8
CH921: Biophysical Techniques
Basic Lab Skills: Nov/Dec 2008
BASIC LABORATORY SKILLS
Introduction. Molecular biology, biochemistry and analytical chemistry all require a very high
standard of basic laboratory skills if reagents and instrument time are not to be wasted. It is
generally assumed that the limiting aspect of an analysis is the equipment used not the operator’s
laboratory expertise. However, it is often the other way round. This session is designed to ensure
you are not the limiting factor. Do not assume that because you have used a particular technique in
an undergraduate course that you are sufficiently proficient. You will need to be checked off at each
stage as indicated. A demonstrator must be convinced you have satisfactorily completed each task.
You will have to have this laboratory session signed off before starting the next laboratory sessions.
Aims. The aims of this excercise are to ensure competency in the use of a 4 figure balance,
calibration and use of micropipette, glass volumetric pipette, graduated glass pipette, measuring
cylinder, and volumetric flask. Glassware is more expensive than you probably realize  so take
care.
You will need to bring to the laboratory:
 A4 laboratory notebook (get from chemistry stores)
 pocket sized notebook
 laboratory coat
 permanent OHP marker pen (for glassware, from chemistry stores)
 normal pen
 safety glasses (from chemistry stores)
You also must prepare safety data forms (before coming to the laboratory) for all compounds used.
YOU ARE NOW RESPONSIBLE FOR THIS. You must read the chemistry department safety
booklet before coming to the laboratory.
Part A. Use of 4-figure balance. Ask a demonstrator to show you how to use, calibrate, tare etc. a
balance.
Part B. Calibration of pipettes and volumetric glassware.
Micropipette. Some of the micropipettes have been recently calibrated and are within 12%, others have not, so this is a dual function exercise. Select a P1000, a P200 or P100, a
P20 or a P2. Examine each pipette and familiarize yourself with its mode of action. Each
student must use 3 pipettes. Read the pipette manual before starting. To set a volume, wind
to a slightly higher value than you want then wind down to the required value. If you
overshoot, rewind to a higher value etc. Make sure the tip is firmly attached. Depress the
plunger to the first stop, immerse tip of tip below the liquid surface (but not right to the
bottom) and slowly suck up liquid (if you get air bubbles in it eject and start again). Remove
the pipette from the liquid. You have now wetted the tip. It is best practice to eject this
aliquot either to waste or back into the sample (consider waste versus contamination issues)
by pressing the plunger to the second stop and repeat the sucking process. Look how much
liquid is in your tip (your eye is surprisingly accurate). Eject the liquid into a container, if
possible with the tip of the tip against the side of the container and the pipette held vertical.
Repeat this a few times to practise. NEVER USE A MICROPIPETTE FOR ANYTHING
EXCEPT water, alcohols (but check calibration), acetonitrile and innocuous aqueous buffer
solutions. Check.
9
CH921: Biophysical Techniques
Basic Lab Skills: Nov/Dec 2008
Calibrate the pipette (and your pipetting) by taking a beaker, placing it on the balance, taring
the balance, and pipetting 1000 µL or 100 µL etc., as appropriate, of water into the beaker.
Record the mass of water in your lab book. Repeat this process until you have 10 successive
aliquots of exactly (to within the pipette's specifications) the same mass each time. Then retare and pipette 10 aliquots into it in quick succession. Weigh the total and determine
whether the pipette is accurate or not. CHECK YOUR PIPETTING METHOD WITH A
DEMONSTRATOR AND GET SIGNED OFF. Check.
Prepare an Excel spreadsheet showing the mean, standard deviation and relative standard
deviation of your 10 individual aliquots for each pipette. Also note the mass of the 10×
aliquots.
Volumetric pipette. Repeat the above with one glass pipettes using a pipette filler. THE
VALVES ON THE FILLERS ARE DELICATE — TAKE CARE. Check with a
demonstrator that you know what you are doing. Note. The pipettes are calibrated to have
the bottom of the meniscus on the line of the pipette. Check.
Graduated pipette. Ensure you understand the markings on a graduated glass pipette.
Measuring cylinder. Repeat the above for a measuring cylinder.
Volumetric flask. Repeat the above for a volumetric flask.
10
CH921: Biophysical Technique
Introduction to Protein Databases Workshop Nov/Dec 2008
INTRODUCTION TO PROTEIN DATA BASES
SWISS PROT: Swiss Prot is a protein sequence database that was established in 1986 and is
maintained by the Department of Medical Biochemistry at the University of Geneva and the
European Bioinformatics Institute, EMBL data library.
Swiss Prot 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 Swiss Prot use the following website link to the Sequence Retrieval System
homepage:
http://us.expasy.org/srs5/

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


Start a new SRS session
Select SWISS PROT (and deselect TREMBL if it is ticked) and press continue
In order to search the database it is necessary to define some fields. It is useful to supply
as much information as is known about the protein. For example searching under
Lysozyme alone reveals 121 hits.
Useful fields to complete include:
Description; Organism from which the protein is from; Keywords or all text.
From the search results choose the most suitable hit, by selecting this general
information can be found.
The primary accession number is a number that will never change, so this can be
transferred to different databases.
ProtParam. To obtain data on chemical and physical properties for a given protein found
using SWISS PROT, a tool called the ProtParam tool can be used. This can be accessed by
using the following website:
http://us.expasy.org/tools/protparam.html
By entering the primary accession number the corresponding proteins sequence will appear.
From this select the region that is the main chain. 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.
Protein DataBank. For information on the structure of a protein use the Protein Data Bank,
web link:
http://www.rcsb.org/pdb/

Once again enter the primary accession number and select find a structure. Many
structures may appear, select the one with the highest resolution, and click on explore.
Record the PDB code, this is the four digit code at the start of each entry.
11
CH921: Biophysical Technique


Introduction to Protein Databases Workshop Nov/Dec 2008
A sequence details page appears, from this display the file by selecting
Download/Display file. 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.
Using this database it is possible to obtain sequence details and view the proteins
structure.
PDBSum. Another useful database for viewing a proteins structure is:
http://www.biochem.ucl.ac.uk/bsm/pdbsum/

Enter the PDB code to view the structure.
12
CH921: Biophysical Techniques
Dichroweb CD Data Fitting Workshop: Nov/Dec 2008
DICHROWEB
Dichroweb is an online circular dichroism analysis facility.
www.cryst.bbk.ac.uk/cdweb

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
Select START ANALYSIS
Login
Enter file information.
The file format is Jasco 1.50 if using data as a .txt file directly from the CD machine, if
converted to delta epsilon use free format (the answer you get ought to be the same!).
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 and A280
Mean residue weight can also be calculated using the molecular weight and number of
residues.
Work to do for each protein.
 From the protein databases find out the extinction coefficent; molecular weight; number
of residues and the protein sequence.
 Use dichroweb to generate plots using ONE fitting program e.g. SELCON.
 In 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):
dE=dA/(c*l)
conc/mg/mL=
pathlength/cm =
0.009185
MW=
concentration/uMaa=
0.0069763 no.aa=
CD/mdeg
Wavelength CD/mdeg zeroed
260 0.0983666 0.0135253
259
0.13072 0.0458787
258 0.070691
-0.01415
0.744
27195 Da
255
HT
delta A
delta E
223.134
4.1E-07 0.0064002
223.398
1.39E-06 0.0217099
224.001
-4.3E-07 -0.006696
13
257
256
255
254
253
252
251
250
249
248
247
246
245
244
243
242
241
240
239
238
237
236
235
234
233
232
231
230
229
228
227
226
225
224
223
222
221
220
219
218
217
216
215
214
213
212
211
210
209
208
0.0415099
0.0667098
0.103352
0.103459
0.052049
0.0608287
0.0720854
0.104696
0.091961
0.0626229
0.0540286
-0.011942
-0.080257
-0.026234
-0.008666
-0.109426
-0.133588
-0.129282
-0.108148
-0.208626
-0.243206
-0.326162
-0.344105
-0.356551
-0.587904
-0.72738
-0.683335
-0.97801
-1.19698
-1.38729
-1.6364
-1.68129
-1.87631
-2.092
-2.36917
-2.57345
-2.73054
-2.83816
-3.04081
-3.12965
-3.14932
-3.13956
-3.21076
-3.13064
-3.02681
-2.94559
-2.75135
-2.66393
-2.4253
-2.14786
-0.043331
-0.018131
0.0185107
0.0186177
-0.032792
-0.024013
-0.012756
0.0198547
0.0071197
-0.022218
-0.030813
-0.096783
-0.165098
-0.111075
-0.093507
-0.194267
-0.218429
-0.214123
-0.192989
-0.293467
-0.328047
-0.411003
-0.428946
-0.441392
-0.672745
-0.812221
-0.768176
-1.062851
-1.281821
-1.472131
-1.721241
-1.766131
-1.961151
-2.176841
-2.454011
-2.658291
-2.815381
-2.923001
-3.125651
-3.214491
-3.234161
-3.224401
-3.295601
-3.215481
-3.111651
-3.030431
-2.836191
-2.748771
-2.510141
-2.232701
224.634
225.243
225.838
226.454
227.06
227.666
228.295
228.938
229.573
230.22
230.894
231.606
232.361
233.15
234.002
234.962
236.02
237.199
238.525
239.958
241.459
243.029
244.669
246.382
248.138
249.92
251.698
253.46
255.201
256.915
258.533
260.075
261.578
263.048
264.513
265.992
267.472
268.95
270.407
271.888
273.436
275.079
276.821
278.656
280.683
283
285.571
288.438
291.636
295.25
14
-1.3E-06
-5.5E-07
5.61E-07
5.65E-07
-9.9E-07
-7.3E-07
-3.9E-07
6.02E-07
2.16E-07
-6.7E-07
-9.3E-07
-2.9E-06
-5E-06
-3.4E-06
-2.8E-06
-5.9E-06
-6.6E-06
-6.5E-06
-5.9E-06
-8.9E-06
-9.9E-06
-1.2E-05
-1.3E-05
-1.3E-05
-2E-05
-2.5E-05
-2.3E-05
-3.2E-05
-3.9E-05
-4.5E-05
-5.2E-05
-5.4E-05
-5.9E-05
-6.6E-05
-7.4E-05
-8.1E-05
-8.5E-05
-8.9E-05
-9.5E-05
-9.7E-05
-9.8E-05
-9.8E-05
-1E-04
-9.7E-05
-9.4E-05
-9.2E-05
-8.6E-05
-8.3E-05
-7.6E-05
-6.8E-05
-0.020504
-0.00858
0.0087593
0.0088099
-0.015517
-0.011363
-0.006036
0.0093953
0.0033691
-0.010514
-0.014581
-0.045798
-0.078125
-0.052561
-0.044248
-0.091928
-0.103361
-0.101323
-0.091323
-0.138869
-0.155232
-0.194487
-0.202978
-0.208868
-0.318344
-0.384344
-0.363502
-0.502943
-0.60656
-0.696615
-0.814494
-0.835736
-0.92802
-1.030085
-1.161242
-1.257907
-1.332243
-1.383169
-1.479063
-1.521102
-1.53041
-1.525792
-1.559484
-1.521571
-1.472438
-1.434005
-1.34209
-1.300723
-1.187803
-1.056518
207
206
205
204
203
202
201
200
199
198
197
196
195
194
193
192
191
190
-1.97766
-1.72507
-1.3651
-1.00368
-0.59642
-0.126838
0.295065
0.537299
0.840225
1.11442
1.57238
2.41985
2.86705
2.56803
2.78953
3.23624
2.558
2.68676
-2.062501
-1.809911
-1.449941
-1.088521
-0.681261
-0.211679
0.2102237
0.4524577
0.7553837
1.0295787
1.4875387
2.3350087
2.7822087
2.4831887
2.7046887
3.1513987
2.4731587
2.6019187
299.33
303.87
308.96
314.671
320.966
327.857
335.361
343.46
351.963
360.738
369.644
378.579
387.588
396.939
406.767
416.093
426.171
436.241
15
-6.3E-05
-5.5E-05
-4.4E-05
-3.3E-05
-2.1E-05
-6.4E-06
6.37E-06
1.37E-05
2.29E-05
3.12E-05
4.51E-05
7.08E-05
8.44E-05
7.53E-05
8.2E-05
9.56E-05
7.5E-05
7.89E-05
-0.975979
-0.856453
-0.686114
-0.51509
-0.322374
-0.100167
0.0994782
0.2141037
0.3574487
0.4871982
0.7039056
1.1049297
1.3165454
1.1750487
1.2798628
1.4912467
1.1703025
1.2312319
CH921: Biophysical Techniques
Experiment I: Nov/Dec 2008
EXPERIMENT I:
REAGENT AND BUFFER PREPARATION
Reagent preparation. You will be allocated solutions from the following list to make up.
Determine which experiment the reagent is used in (you will have to read ahead in your lab
manuals) and calculate how much of the reagent each person will require. To determine how much
total solution to make, multiply by a factor of class number times 1.25.
1.1
200 M [Ru(1,10-phenanthroline)3]2+ in water
1.2
100 mM NaCl in water
1.3
50 mM phosphate buffer, pH=7. (4 people) (NB don’t waste solutions  you will need to
make up much more of one component than the other)
1.4
2 mg/mL protein standard solution
1.5
Biuret reagent: Place CuSO4.5H2O (1.5 g) and sodium potassium tartrate.4H20 (6.0 g) into a
dry 1 L volumetric flask add about 500 cm3 of water. With constant swirling, add NaOH
solution (300 mL, 10% w/v). Make to volume (1L) and mix. The reagent prepared in this
manner is a deep blue. It may be stored indefinitely if KI (1 g) is also added and the reagent
is kept in a plastic container.
1.6
Dissolve Coomassie Brilliant Blue G-250 (100 mg) in 50 mL 95% ethanol. To this solution
phosphoric acid (100 cm3 85% w/v) is added and the solution diluted to 1 L. (Alternatively,
a pre-prepared dye reagent concentrate can be purchased from Bio-Rad and diluted by
adding 4 volumes of distilled water to 1 volume of concentrate.)
Buffers. The following tables describe how two important buffers, acetate and phosphate buffer,
are prepared at a range of pH values by mixing different amounts of two stock solutions. (From
Methods in Enzymology, Vol. 1, p.138)
Acetate buffer. Stock solutions:
A: 0.2 M solution of acetic acid (11.55 mL in 1000 mL H2O)
B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or
27.2 g of C2H3O2Na.3H2O in 1000 mL.
(mL A + mL B, diluted to a total of 100 mL)
A (mL)
46.3
44.0
41.0
36.8
30.5
25.5
B (mL)
3.7
6.0
9.0
13.2
19.5
24.5
pH
3.6
3.8
4.0
4.2
4.4
4.6
A (mL)
20.0
14.8
10.5
8.8
4.8
16
B (mL)
30.0
35.2
39.5
41.2
45.2
pH
4.8
5.0
5.2
5.4
5.6
CH921: Biophysical Techniques
Experiment I: Nov/Dec 2008
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).
A (mL)
93.5
92.0
90.0
87.7
85.0
81.5
77.5
73.5
68.5
62.5
56.5
51.0
(mL A + mL B, diluted to a total of 200 mL)
B (mL)
pH
A (mL)
B (mL)
6.5
5.7
45.0
55.0
8.0
5.8
39.0
61.0
10.0
5.9
33.0
67.0
12.3
6.0
28.0
72.0
15.0
6.1
23.0
77.0
18.5
6.2
19.0
81.0
22.5
6.3
16.0
84.0
26.5
6.4
13.0
87.0
31.5
6.5
10.5
90.5
37.5
6.6
8.5
91.5
43.5
6.7
7.0
93.0
49.0
6.8
5.3
94.7
pH
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
Questions (answers to be submitted).
1. How does a buffer work? What determines its pH range?
2. Why might you need to use acetate rather than phosphate?
3. What is the concentration of sodium in the standard pH = 7.2, pH = 7.0 and pH = 6.8 phosphate
buffer?
4. Write brief notes on buffers. Consider a pH=7 buffer. If 1 mL of a 100 mM (in phosphate) stock
solution is used, how much buffering can this solution do? Is it likely to be enough for a solution of
1 mL of 2 mg/mL protein?
17
CH921: Biophysical Techniques
Experiment II: Nov/Dec 2008
EXPERIMENT II:
PREPARATION OF DNA AND PROTEIN SAMPLES
Aim. The aim of this experiment is to prepare the DNA and proteins samples which you will use in
Experiments 3-5. Before weighing out your DNA or protein you must read ahead in the lab script
and calculate the amounts of these substances you will need (this is discussed in more detail in Parts
A and B).
Part A: DNA Sample preparation. Determine how much DNA you require to make up 5 mL of a
1400 M in base DNA solution. (Take a base to have average molecular weight of ~330
Da.) Get this checked with a demonstrator. Once approved, weigh out this amount of calf
thymus DNA and add 4 mL of water. Make sure the DNA is beginning to hydrate (it will
start to look translucent) then leave it in the refrigerator over night. Calf thymus DNA is
sufficiently stable that we can get away without buffering it for this experiment.
Part B: Protein sample preparation. You will be making up your protein sample (US) to be ~ 1
mg/mL. But note that this will not be an accurate guide to concentration as it may not be
pure but contaminated with salts or nucleic acids. Determine how much stock solution you
will need to run a circular dichroism spectrum (200 L of 0.1 mg/mL and 60 L of 1
mg/mL) and to do the required protein concentration determination experiments. Construct a
table with the required volumes of 1 mg/mL stock needed for each assay, and get this
checked with a demonstrator. Once approved, make up this solution in water. If the protein
is reluctant to dissolve you may need to add a drop of dilute (~ 1 M or less) acid (usually
one chooses HCl).
Protein List:
 Lysozyme
 Ribonuclease A
 -Lactalbumin
 Myoglobin
 -Chymotrypsin
 Cytochrome c
18
CH921: Biophysical Techniques
Experiment III: Nov/Dec 2008
EXPERIMENT III:
COMPARISON OF THREE METHODS FOR THE ESTIMATION OF PROTEIN
CONCENTRATION
Aims. For a variety of purposes, including all structural studies of proteins and in order to
determine the specific activity of an enzyme at different stages of purification, one must
have a sensitive method for estimating protein concentration. In this experiment you will
compare three different methods and evaluate their relative merits.
Solutions. At this point, you will have two protein stock solutions which you will use
throughout Experiment III. The first is the protein stock solution you made in Experiment
II, referred to here as unknown protein stock (or US) and having a concentration of
approximately 1 mg/mL. The second protein stock solution is that of a protein standard
stock (denoted SS), whose concentration is accurately known and given on the bottle (~2
mg / mL). With each method you will need to dilute the stock to the appropriate
concentration range for the assay. Note in your laboratory book and your report what
(weighing, measuring volumes etc. in a table) you have done to make the solutions you
have used. In each case perform repeat measurements on each unknown sample.
Calibration curves. For Assays B, C, and D, you will need to plot calibration curves using
results from a protein of known concentration (made from SS). Plot absorbance verses µg of
protein in the assay mixture. You may plot the data electronically but a plot on graph
paper will be required for your assessment. Use your curve to determine the µg of U in
your assay mixtures by drawing a horizontal line from the absorbance reading of the
unknown to the calibration curve, then dropping a vertical line to read the µg of protein in
the mixture. Hence determine the concentrations in the stock solutions.
Assessment. In addition to standard requirement, please include the following in your
report.
 Plot standard curves using the data for the known concentrations. Determine the U
concentrations using each method. Discuss your results in terms of the relative sensitivity
and accuracy of the four methods. Comment on the errors in the measurements.
 Outline the chemistry of each method.
19
CH921: Biophysical Techniques
Experiment III-A: Nov/Dec 2008
ASSAY A: ABSORBANCE AT 280 NM.
In this assay you will measure the A280 of the approximately 1 mg/mL US protein sample in
quartz cuvettes. You will then use this information, along with the extinction coefficient
determined in the data base exercise, to calculate concentration.
Protocol. Put ~ 2 mL 18.2 M water in either a clean dry or clean water rinsed quartz
absorbance cuvette. Set the instrument parameters (in file menu on V-550 or V-570) to
collect data from 400 - 200 nm in 0.5 nm steps with a response time of ~ 400 nm/min (fast).
Run a baseline/background spectrum with the cuvette in the sample position.
Take a clean dry 1 cm quartz absorbance cuvette (you may need to wash an old sample out
by emptying the cuvette, filling it with water, emptying it - repeat at least 2 times). Then fill
with acetone, empty, repeat 2 times. Dry cuvette either with nitrogen line (or air if nitrogen
is not available) or a hair dryer. Pipette directly into the cuvette: 1 mL of your US; add 1
mL 18.2 M water.
On the V-550/570 select autozero from the file menu. Measure a spectrum using the same
parameters as the baseline. Save your data directly onto a floppy disk as jws format and txt
format if you are using the V-550/570.
Determine  for your protein from its amino acid sequence (see computer session for how to
get this information or calculate it from the sequence as indicated in lectures)*. Use the
Beer Lambert Law to determine the concentration of your US. Compare this value with that
obtained by assuming that a 1 mg/mL solution has an absorbance of 1.0. Comment on any
differences. What is the assumption underlying this method? Compare both values for
concentration with that obtained from the equation: 1.55  A280 - 0.76  A260 = mg
protein/mL. Comment. What is the rationale behind this equation? (*cystine, max~120
mol1dm3cm1; Tyr, max ~ 1280 mol1dm3cm1; Trp, max~ 5690).
Exercise. If lysozyme has a molecular weight of 14314, nW = 6, nY = 3, nC = 8, determine
its . Compare this with the experimental value of 280 = 37932 mol1dm3cm1. The values
for chymotrysinogen are: 25670, 8, 4, 10, 51340. Comment.
20
CH921: Biophysical Techniques
Experiment III-B: Nov/Dec 2008
ASSAY B: BIURET METHOD.
(Reference: Gornall, Bardawils and David, J Biol Chem 1949, 177, 751)
This method is simple and reasonably specific as it depends on the reaction of copper (II)
with N atoms in the peptide bonds of proteins. Compounds containing peptide bonds give a
characteristic purple colour when treated in alkaline solution with copper sulfate. This is
termed the 'biuret' reaction because it is also given by the substance biuret NH2—CO—NH
CO—NH2, a simple model compound.
H
O
R
H
O
N
C
C
H
N
C
Cu(II)
C
N
CH
C
N
O
H
R
O
H
For a wide variety of proteins, 1.0 mg of protein in 2 mL of solution results in an OD at 540
nm of 0.100. This assay is sensitive to 0.5 – 2.5 mg protein in the assay mixture
Many haemoproteins give spurious results due to their intrinsic absorption at 540 nm, but
modifications which overcome this difficulty are known (either removal of the haem before
protein estimation or destruction of the haem by hydrogen peroxide treatment). The protein
content of cell fractions such as nucleii and microsomes can be estimated by this method
after solubilisation by detergents such as deoxycholate or sodium dodecyl sulphate.
Protocol. You will begin by preparing SS standards for a calibration curve containing: 0.0
mg protein; 0.5 mg protein (e.g. 250 µL SS solution); 1.0 mg protein; 1.5 mg protein; and 2
mg protein from the 2 mg / mL SS protein solution provided. Also prepare duplicates of 2
different concentrations of US (US should always be measured in duplicate). To prepare
the protein solutions, mix the protein solution (x µL, where x < 1500 µL) with water [(1500
- x) µL] to make a total volume of 1500 µL. Summarize your calculations in a table and
have it checked by a demonstrator. Once approved, prepare your samples.
Add 1500 µL biuret reagent and mix. The purple colour is developed by incubating for 15
minutes at 37C. Cool the tubes rapidly to room temperature. Measure a spectrum of this
solution and also of a reference solution containing 1500 µL biuret reagent and 1500 µL
water, which has also been incubated at 37C. Samples to be measured should be at room
temperature and should not be unduly warm or ice cold because the colour intensity of the
copper complex has a high temperature coefficient. Read the absorbances at 540 nm. The
colour of the solutions is stable for hours. Plot a calibration curve using protein standard
and use the curve to determine the concentration of US. * Important note: the BeerLambert Law does not hold for these solutions at optical densities above 0.25.
Relatively few substances interfere with the biuret estimation; those which do, include bile
pigments, sucrose, tris, glycerol, imidazole and ammonium ions. Sucrose, tris and glycerol
can usually be corrected for by their inclusion in the blank and protein standard.
21
CH921: Biophysical Techniques
Experiment III-C: Nov/Dec 2008
ASSAY C: COOMASSIE BLUE DYE BINDING ASSAY.
(References: MM Bradford, Analytical Biochemistry 1976, 72, 248; SM Read, DH
Northcliffe, Anal Biochem 1981, 96, 53.)
This protein determination method involves the binding of Coomassie Brilliant Blue G250
to protein. The protonated form of Coomassie Blue is a pale orange-red colour whereas the
unprotonated form is blue.
When proteins bind Coomassie Blue in acid solution their positive charges suppress the
protonation and a blue colour results. It has been found that hydrophobic interactions
between the dye and the protein are very important in the binding process. The binding of
the dye to a protein causes a shift in the absorption maximum of the dye from 465 to 595
nm and it is the increase in absorbance at 595 nm which is monitored. The assay is very
reproducible and rapid with the dye binding process virtually complete in ~ 2 minutes with
good colour stability.
The only compounds found to give excess interfering colour in the assay are relatively large
amounts of detergents such as sodium dodecyl sulphate, Triton X-100 and commercial
glassware detergents. Interference by small amounts of detergent may be eliminated by the
use of proper controls. The assay is non-linear and requires a standard curve. The standard
assay described below is useful for protein solutions containing 10 to 100 µg of protein in a
volume up to 100 µL. (The micro-protein assay described in Bradford's article can be used
for protein solutions containing 1 to 10 µg proteins in a volume up to 100 µL, but requires
the use of a microcuvette.)
Protocol. You will again begin by preparing SS standards for a calibration curve
containing: 0 µg, 20 µg, 40 µg, 60 µg, 100 µg of protein from the 2 mg / mL SS protein
solution provided. Also prepare duplicates of 2 different concentrations of US. Because this
assay is very sensitive (sensitivity 20 – 140 µg protein), you will need to prepare more
dilute (1/5 th concentration) standard solutions from SS and US.
Place required volumes, x µL, of the 1/5 th concentration standard solutions in clean, dry
test tubes. Add (500 – x) µL water. Also place 500 µL water or sample buffer in "blank"
test tubes. Add 5.0 mL diluted dye reagent (Bio-Rad) to each sample. Vortex (avoid excess
foaming) or mix several times by gentle inversion of test tube. After a period of from 5 –
60 minutes, determinine A595. Plot A595 versus the amount of protein in each assay tube.
Read unknowns from the standard curve.
22
CH921: Biophysical Techniques
Experiment III-D: Nov/Dec 2008
ASSAY D: BCA METHOD
A standard solution of a chosen standard protein (usually 2.00 mg/mL bovine serum
albumin) should be created. In this case the table below summarises the protein solutions
that should be made for the calibration curve. Two U sample concentrations should be
chosen to give values in the middle of the calibration graph. Repeat the measurement of
each U concentration.
Protein concentration
(g/mL)
1000
500
200
50
0
L 2 mg/mL BSA
solution
50
25
10
2.5
0
L buffer or water
50
75
90
97.5
100
Protocol. Make a stock reaction mixture solution by mixing 20 mL BCA reagent, Pierce
No. 23223 and 285 L 4% CuSO4. For each analysis: make 100 L protein solution of the
desired concentration in buffer or water. Mix well. Add 2 mL of the reaction mixture from
step 1. Mix well. Incubate at 37C for 30 minutes.
Allow the tubes to cool down to room temperature and then measure the absorbance at 562
nm having zeroed the spectrometer on a water sample. Plot the readings for each standard
as a function of protein concentration. Use the resulting curve to determine the concentration
of the unknown protein.
23
CH921: Biophysical Techniques
Experiment IV: Nov/Dec 2008
EXPERIMENT IV:
PROTEIN SECONDARY STRUCTURE DETERMINATION BY CIRCULAR DICHROISM
Protocol. The practical part of this experiment is straight forward and requires a 0.1
mg/mL solution of your US protein to be put into a 1 mm quarz cuvette and a CD
spectrum collected from 260 – 190 nm. A buffer baseline (which will already have been
run by the demonstrator) needs to be subtracted to give you the sample’s CD spectrum.
Repeat the experiment with 1 mg/mL US protein solution and a 0.1 mm demountable cell.
Parameters should be: 100 nm/s; response time = 1 s; data interval = 1 nm; bandwidth = 2
nm; accumulations = 4. Wash your cuvette with water (at least 3 times) and acetone (3
times). Dry it with a hair dryer or nitrogen line.
You should save your data files (sample and baseline) as a txt files for analysis. Use Excel
to subtract the baselines and plot the CD spectra of the proteins (both in mdeg and ).
You will need in addition to your spectrum a reasonably accurate molar concentration of
your protein solution. Ideally this will come from Experiment III or Experiment III as
revised during the analysis session. Determine the -helical content of your protein as
given below and compare the answer with that from the crystallographic data base.
IAMBEC: assume that 100% -helical protein has 208 nm ~ -12 mol-1 dm3 cm-1.
MOAC: Use Curtis Johnson’s CD structure fitting program CDsstr.
CD structure fitting data analysis using CDsstr for far UV spectra. For each sample
that has been measured for which CD structure fitting is required, take the text file for the
baseline substracted and zeroed spectrum and edit it in Excel or another piece of software
to produce the data in the following form: One title line containing anything, followed by
71 lines (assuming fitting is being undertaken from 260 nm to 190 nm) of numbers
corresponding to the CD spectrum in units of moles of (amino acids)-1 dm3 cm-1 with only
two decimal places. If you have more than one data set, the second set starts on the line
directly below the first.
nsure that the CDsstr program and the required associated files are located in a directory
on the C drive of the computer you are using. The files include: procd190.tst; readme.cd;
secstr.dta; bascd.dta; Cdsstr.exe. procd190.tst is a data file that can be used to test the
program; it has three data sets in it.
To run the program, proceed as follows. Delete, rename, or move any file with a .out
filename extension remaining in the CDsstr folder. Delete any previously used file named
proCD.dta unless you wish to use it in the current run. If it is not already available,
prepare an input file called procd.dta containing the CD data of the protein(s) to be
analyzed. Save the file as c:\cdsstr\proCD.dta. Begin the analysis by opening a DOS
window within windows. Type ‘c:’. Then type ‘cd\cdsstr’ at the command prompt. Type
‘cdsstr’ to run the program. Enter values for the program variables as prompted. NbasCD
= 22; Nwave = 71; Npro = number of data sets in procd.dta; ncomb = 100; icombf =
100000. When the command prompt reappears, view, print and record in the laboratory
book the results of the analysis by inspecting the output files anal.out and reconCD.out.
24
CH921: Biophysical Techniques
Experiment V: Nov/Dec 2008
EXPERIMENT V:
EQUILIBRIUM BINDING CONSTANT DETERMINATION BY FLUORESCENCE
SPECTROSCOPY: DNA AND [RU(1,10-PHENANTROLINE)3]2+
This experiment is referred to as a titration series but you will save instrument time by
preparing all the solutions before hand. (If the DNA is very expensive then you can save
sample by adding DNA successively to the cuvette and simultaneously adding the same
volume of ligand at twice the cuvette concentration. Alternatively you can dilute a
concentrated DNA cuvette by adding ligand at the appropriate concentration.) Each
solution will have 10 M of Ru and a varying amount of DNA: 0, 10 M, 20 M, 40 M,
80 M and 300 M.
Protocol. Determine the concentration of your DNA stock solution by measuring the A260
for a ~100 M DNA in base solution by measuring the absorbance spectrum from 320 200 nm. 258(calf thymus DNA) = 6600 mol-1 dm3 cm-1. Repeat the measurement (at least
twice) until a consistent value is obtained.
Construct a table of volumes of stock solutions (including water) required to enable you to
run the experiment at the above DNA and Ru concentrations with 10 mM NaCl and 1 mM
(pH=7) phosphate buffer. Total volumes should be 3 mL. Check your table with a
demonstrator.
Make up one Ru solution, 5 containing Ru and DNA and one containing only buffer and
water. Collect fluorescence spectra for the seven samples. Save the data to disk as txt
files. Determine the concentrations of bound and free Ru in each case and use the data to
perform a Scatchard plot as outlined in lectures.
25
CH921: Biophysical Techniques
DNA Melting Curve Exercise: Nov/Dec 2008
DNA MELTING CURVE. Determination of thermodynamics of the helix to coil transition
INTRODUCTION. Under normal physiological conditions DNA exists as a double helix with two
polymers held together by hydrogen bonds between the bases on each strand. Most biological
processes involving DNA (including transcription, replication) require the DNA to separate into
single stranded components. We can model this process to determine aspects of its thermodynamics
by taking a solution of double stranded (ds) DNA helices and gradually heating them up until each
DNA duplex has separated into two single stranded (ss) pieces of DNA that are often described as
random coils. The so-called melting temperature of a DNA helix, Tm, is defined to be the
temperature at which half the DNA base pairs have broken their hydrogen bonds. As the melting is
cooperative (a single molecule unzips once it has started) this is usually equivalent to half the DNA
molecules being ds and half ss.
Duplex DNA has the DNA bases held together in a fairly rigid arrangement with each base stacked
between the bases above it and below it and hydrogen bonded to a base in the other strand. When
the ds to ss transition takes place, the bases move much more freely and the base-base stacking is
essentially removed. This means the π electrons of one base are no longer interacting strongly with
those of the neighbouring base. The removal of π-π stacking causes the UV absorbance of the bases
at about 260 nm to increase in magnitude. We can therefore monitor the transition by measuring the
absorbance of a DNA sample as a function of temperature. You will have two columns of data. The
first column is the temperature at which a measurement is taken, T, and the second column is the
absorbance of a piece of DNA at that temperature, A. The following series of calculations will
enable you to determine H, S, and G for the helix to coil transition.
1.
2.
3.
4.
Plot A versus T. (i.e. take A as the y axis and T as the x-axis). Estimate Tm by using a ruler to
determine the point half way between the low T A curve and the high T curve. The value you
determine is only approximate if the baseline of the curve is not flat.
Differentiate the A curve with respect to T. The maximum of this plot is another approximate
value for Tm.
Using a ruler draw a straight line through the first 15 degrees of data points. This line
approximates the temperature dependence of the unmelted DNA absorbance if you could hold
it together. Work out an equation for this line and use this to create a column of data points that
plot the straight line overlaid on top of your part 1 curve.
Determine the final absorbance on the melting curve plot, then evaluate

5.
6.
x
xy

as a function of temperature where x is the difference between the final absorbance of the
unfolded DNA and the absorbance at a given temperature and y is the difference in absorbance
between the temperature dependent absorbance and the dotted base line of your part 3 figure.
Create a temperature column in Kelvin, Tabs, and then create a 1/Tabs column. Plot  versus
the inverse of the absolute temperature (in Kelvin). The melting temperature is then the
temperature at which half of the DNA is melted, i.e. the midpoint of the  versus 1/T curve.
Plot the derivative of  with respect to the inverse of the absolute temperature versus T.
Determine the van't Hoff transition enthalpy of the transition
26
HVH 
B'

(1 / T max)  (1/ T2 )
where Tmax is the absolute temperature of the maximum of the derivative curve you have just
plotted and T2 is the absolute temperature of the high temperature half height of the curve. B'
=  4.38 cal K1 mol1; to convert from calories to Joules multiply by 4.1868 J/cal.
7.
Determine the entropy change of the transition using
S 
8.
HVH

Tm
Determine the Gibbs free energy of the transition and comment on the magnitudes of the three
thermodynamic properties you have determined.
Reference
L.A. Marky; K.J. Breslauer, Biopolymers, 1987, 26, 1601–1620
27