___________________________________________________________________________
MOAC D O C T O R A L T R A I N I N G C E N T R E
N O V E M B E R /D E C E M B E R 2013
CH921:
TECHNIQUES FOR THE CHARACTERISATION OF
BIOMOLECULES
COURSE LEADER: DR. ANN DIXON
Contributors: Prof. Alison Rodger; Prof. Peter O'Connor, Dr.
Claudia Blindauer; Prof. Steven Brown; Dr. Alexander
Cameron; Dr. Jozef Lewandowski, Dr. Ivan Prokes
TABLE OF CONTENTS
___________________________________________________________________________
PAGE
Deadlines and other important information
3
Timetable
4
Essay assignment
5
Laboratory and workshop manual:
Workshop 1
6-8
Protein List
8
Workshop II
9
Aims and Assessment
10
Guidelines for preparation of lab report
11
Experiment I
12-17
Experiment II
18-22
Experiment III
23-24
2
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 Naomi Grew / Catherine Findley has your correct email address. You will
be notified of timetable changes by email with at least 24 hours notice. Failure to
note timetable changes will result in loss of credit for attendance.

As:MIT students to submit all work to Christina Forbes.

MOAC students to submit all work to Naomi Grew / Catherine Findley.

Plagiarism policy: Any text directly cut and pasted from the internet or any online or
electronic source will be automatically regarded as plagiarism. In cases where a
particular phrase is reproduced directly from a published source (of any type), then
the source should be referenced in full at the point at which it is quoted. Furthermore,
the amount of directly reproduced phrases should be minimal and limited to what is
essential to support the arguments presented in the text. In any case the total amount
of directly reproduced (and referenced) phrases should not exceed 5% of the full piece
of work. Complex diagrams, which would otherwise be difficult to reproduce, may be
taken from a published source provided that the source is directly referenced and the
appropriate reproduction permission has been achieved, if required (not needed for
essays or laboratory reports).
ASSESSED WORK AND DEADLINES
___________________________________________________________________________
Deadlines are serious. Marks will be deducted from late work (1% / hour late; 5%/day
late) unless an extension is granted in writing from Dr. A. Dixon.

Workshop problems/proof of completion:



Essay:
NMR assessment:
Laboratory reports:
Due by 5 pm on the day of the
workshop.
Due Tuesday 19 November at 4 pm.
Due Tuesday 3 December at 4 pm.
Due Tuesday 10 December by 4 pm.
An oral examination (with 2 hours written/reading work before hand, to be submitted at
the oral) will take place on Monday 2 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 TIMETABLE AUTUMN 2013
___________________________________________________________________________
WEEK 6:
Mon.
9:00-10:00:
Introduction to techniques for the characterization of biomolecules
Nov. 4
(Dixon, MOAC Lecture Room)
10:00-12:00: Databases workshop (Dixon, MOAC Lecture Room)
13:00-17:00: Obtain Essay title and begin work on Essay
Tues.
Nov. 5
14:00-17:00:
UV lecture and workshop (Rodger, MOAC Lecture Room)
Fluorescence lecture, & workshop, (DNA melting curve) (Rodger,
MOAC Lecture Room)
Laboratory (Dixon, Chemistry B309)
9:00-11:00:
11:00-12:00:
14:00-16:00:
CD lecture (Rodger, MOAC Lecture Room)
CD workshop (Rodger, MOAC Lecture Room)
Linear Dichroism lecture (Rodger, MOAC Lecture Room)
Tues.
Nov. 12
9:00-10:00:
10:00-17:00:
Pre-lab (Dixon, MOAC Lecture Room)
Laboratory (Dixon, Chemistry B309)
WEEK 8:
Mon.
Nov. 18
9:00-12:00:
Introduction to Mass Spectrometry (O'Connor, MOAC Lecture
Room)
WEEK 7:
Mon.
Nov. 11
Tues.
Nov. 19
WEEK 9:
Mon.
Nov. 25
Tues.
Nov. 26
WEEK 10:
Mon.
Dec. 2
Tues.
Dec. 3
WEEK 10+1:
Tues. Dec. 10
9:00-11:00:
11:00-13:00:
9:00-13:00:
Introduction to NMR (Brown, MOAC Lecture Room)
14:00-17:00: NMR Group Work (Brown, MOAC Lecture Room)
*Deadline 1: Essay due (4 pm)
9:00-12:00:
13:00-16:00:
Bio-applications of high field NMR (Blindauer, MOAC Lecture Room)
X-Ray crystallography lecture and demo (Cameron, BSR5, Bio. Sci)
NMR Demonstrations
9:30-12:30 (MOAC Students): (Lewandowski, Milburn House)
10:00-12:00 (As:MIT Students): (Prokes, Milburn House)
9:00-17:00:
EXAM
10:00-11:00: Pre-lab (Dixon, MOAC Lecture Room)
11:00-16:00: Laboratory: (Dixon, Chemistry B309)
*Deadline 2: NMR Assessment due (4 pm)
*Deadline 3: Lab reports & Feedback forms due
4
ESSAY
___________________________________________________________________________
Select one of the following essay topics:
1. The structures of membrane proteins
2. Sugar-binding proteins: Structure and Function
3. The structure of viral particles / viruses
Include in your essay a discussion of the structure of the system you have chosen. Also
include how different biophysical techniques (NMR, X-ray, microscopy, spectroscopy, etc)
have been used to acquire the structural data. Conclude with a critical analysis of the
techniques used including their advantages and disadvantages.
Write using an American Chemical Society Journal template, found at:
http://pubs.acs.org/page/jacsat/submission/jacsat_templates.html
and cite references using an ACS approved style - for a nice summary see:
http://library.williams.edu/citing/styles/acs.php
Use at least 10 primary recent literature references. Web pages are not included as
references in this count but must be acknowledged if you use diagrams taken from
websites.
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, format etc.
Both an electronic and hard copy version should be submitted to Naomi Grew / Catherine
Findley on or before the deadline of 4 pm, Tues. Nov. 19, 2012.
5
WORKSHOP I: INTRODUCTION TO PROTEIN DATA BASES
UNIPROT
___________________________________________________________________________
The Universal Protein Resource (or UniProt) is a comprehensive resource for protein
sequence and annotation data. UniProt consists primarily of three databases: the UniProt
Knowledgebase (UniProtKB); the UniProt Reference Clusters (UniRef); and the UniProt
Archive (UniParc). UniProt is a collaboration between the European Bioinformatics Institute
(EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR)
with the objective of the curation and organisation of huge numbers of protein sequences.
UniProt gives information on the function(s) of proteins, post-translational modification(s),
domains and sites, secondary and quarternary structures, similarities to other proteins,
sequence conflicts and variants, and disease(s) associated with deficiency(s) in the protein.
There is a high level of integration with other biomolecular databases.
To enter UniProt, use the following website link to the Sequence Retrieval System
homepage:
http://www.uniprot.org/







Select “UniProt KB” (UniProt knowledge base) from the “Search in” drop down menu,
but note that there are other options available that you can explore in your own time.
In the “Query” box, type the name of a protein or other keyword. As a practice run,
type “Ubiquitin”, then click “Search”.
You should have about 124,000 hits – that’s a lot of information about Ubiquitin. But
you’ll need to narrow it down as you will most often be looking for information about
a particular protein.
Click the “advanced search” link on the right-hand side of the screen, and type in
another search term to narrow down the number of hits. In this case, we will search
for Ubiquitin from a particular organism. Select “Organism” from the “Field” drop
down menu, but again note that there are other options available that you could use
to narrow your search. Then type “Human”, select from the list, and click “search”.
Now you should have over 13,000 hits – that is still a lot, but in this case the one you
want is the third hit (primary accession number P62979 (RS27A_HUMAN)). Note: The
primary accession number is a number that will not change and is used for only one
protein, and this can be transferred to different databases.
Select RS27A_HUMAN by clicking on the accession number (P62979), and this will
bring up the file on this particular protein. Scroll down and you will see a large variety
of information about this protein, including key references, links and the protein
sequence.
Go to the protein sequence and you will see a drop down menu labelled “Tools”. You
will now use these tools to analyse the protein sequence.
PROTPARAM
___________________________________________________________________________
To obtain data on chemical and physical properties for a given protein found using UniProt,
a tool called ProtParam can be used. This can be accessed by clicking “ProtParam” in the
6
“Tools” drop down menu next to the protein sequence, and then clicking “go”.
Alternatively, you can go directly to the ProtParam analysis website at:
http://expasy.org/tools/protparam.html
and entering either the primary accession number or the sequence of the corresponding
protein.
Once you have clicked “ProtParam” in the “Tools” drop down menu next to your protein
sequence, select the region that is the main chain (1-76 in the case of Ubiquitin). This will
give information about the number of amino acids; molecular weight; amino acid
composition; chemical formula and extinction coefficient with and without disulfide bonds
amongst other information.
RCSB PROTEIN DATA BANK
___________________________________________________________________________
For information on the structure of a protein use the Protein Data Bank, web link:
http://www.pdb.org/pdb/home/home.do




You can search this database using the primary accession number by clicking the
“Advanced Search” link on the upper left hand side of the screen.
In the Advanced Search interface, select “UniProtKB Accession Number(s)” in the
“Choose a query type” drop down menu, and enter your accession number in the
“Accession IDs” box (e.g. P62979). Then select “Submit query”.
You should get approximately 10 hits. If possible, you should look for structures (a) of
just the protein (no bound ligands, etc), and (b) with the highest resolution. For this
example search, you could select the structures with the PDB IDs 2XK5 or 3NOB.
This will connect you to a page with several thumbnails at the top where you can see
structure and sequence details as well as many other types of information. Have a
look around, then view the structure using the .jmol viewer. You can save an image of
the structure by right clicking and selecting the appropriate option for the file type you
prefer. You can also download the structure file by selecting “Download Files”, and
you can display the complete file in text format. With less well known proteins it is
important to search for any missing residues. To do this use find under edit on the
main toolbar and search for MISSING.
YOUR ASSIGNMENT
___________________________________________________________________________
You will be assigned a protein from the list on the following page. Search for and find the
protein you have been assigned in the UniProt database as you just did above. You’ll need
to include the following information in your lab writeup either in the introduction or results
sections:
A. UniProt entry for that protein, including the protein sequence (intro)
B. ProtParam analysis output for the protein (results)
7
C. An image of the X-Ray or NMR structure for that protein (results)
You have the time allocated in the workshop to get feedback on the information you collect
for your protein – this can be tricky so do not leave the workshop until you are sure that you
have selected an appropriate UniProt entry and structure. This information will compose a
portion of the marks in your lab report, and will also be used to help you complete the lab
tasks (you will be using the same protein you were assigned here to perform your lab
experiments), so you’ll need to bring a copy of this information to the lab. Failure to
retrieve the correct information will mean that you’ll have to do the exercise again before
starting the lab experiments (i.e. you’ll have less time in lab).
PROTEIN LIST
___________________________________________________________________________







Lysozyme (chicken egg white)
Albumin (chicken egg white or bovine)
Ribonuclease A (bovine pancreas)
-Lactalbumin
Myoglobin (equine heart)
-Chymotrypsin (bovine pancreas)
Cytochrome c (bovine heart)
8
WORKSHOP II: FITTING OF CIRCULAR DICHROISM DATA
DICHROWEB
___________________________________________________________________________
Dichroweb is an online circular dichroism analysis facility. To access Dichroweb for analysis
of CD data, use the following website link and then follow the instructions below:
www.cryst.bbk.ac.uk/cdweb











Select START ANALYSIS
Login (you will be given a password for this program at the start of the module)
Enter file information.
The file format is Jasco 1.50 if using data as a .txt file directly from the CD machine
(this will be in units of mdeg). If the data has been converted to units of delta epsilon,
use the free format (the answer you get ought to be the same whichever units you
use).
The input units are machine units.
Analysis programs - SELCON and CONTIN may be the best choice.
Reference set — use the best to fit your data.
Output format – default, and output units - machine units.
Submit the form.
Protein concentration can be calculated using (found in the protein databases
workshop) in conjunction with the A280 and Beers Law.
Mean residue weight can also be calculated using the molecular weight and number of
residues.
YOUR ASSIGNMENT FOR EACH PROTEIN
___________________________________________________________________________

From the protein databases, you should determine the extinction coefficient,
molecular weight, number of residues, and the protein sequence.

Use dichroweb to generate plots using ONE fitting program e.g. SELCON.

In MS Excel, plot your original experimental data in machine units and as a delta
epsilon plot (in terms of amino acids). Also plot dichroweb experimental and fitted
data.

Compare your experimental data with the dichroweb data. Record the percentage
secondary structure of your protein.
* Note your username and password here (there will be provided to you):
Username: __________________________________________________
Password: __________________________________________________
* Practice data set (monoclonal antibody fragment) available on CH921 website:
9
LABORATORY PRACTICALS
AIMS
___________________________________________________________________________
The aims of this lab course are to familiarize you with biological samples and teach you
competence in a set of standard spectroscopic and analytical techniques. These include:

COSHH risk assessment, Protein concentration determination (Experiment I).

Determination of protein mass and purity using SDS-PAGE (Experiment II).

Protein secondary structure determination by circular dichroism (Experiment III).
You will be working in pairs in lab, but you will be responsible for independently writing up
your lab reports.
Important: Please read the relevant lab scripts BEFORE coming to lab - you must be
prepared in order to complete experiments in the allocated time. No extra make up session
will be available.
ASSESSMENT
___________________________________________________________________________
Hand in a brief description of the experiments you performed (enough detail so you could
look up your notes during your project and use the techniques), plots of the spectra you
have recorded and the structural deductions you can make from them. Demonstrators will
also be giving you a grade for laboratory work. Aspects being assessed will include:

Improvement of laboratory skills with respect to sample handling

Tidiness and cleanliness

Accuracy of results

Care of equipment

Organization and efficiency in the laboratory.
Your laboratory reports may form the basis of part of your oral examination for this module.
Also include information from the databases about the protein you have worked with
including: molecular weight, amino acid residue content, extinction coefficient, -helix
content as determined from the crystal structure.
BEFORE COMING TO LAB
__________________________________________________________________________

Read lab scripts for the current day.

Perform all required calculations - lab time is limited and you will not be allowed to
stay longer than scheduled session.

Obtain lab coat, safety glasses and lab book and bring ALL THREE to every lab session.
If you do not have all of these items as well as your lab manual, you will be asked to go
get these items.
10
FORMAT FOR LAB WRITEUPS
__________________________________________________________________________
Write up Experiments 1-3 as a single report. Reports should be NO LONGER than 15 pages
with 1.5 spacing between lines and no smaller than 11 point Arial font. Points will be
deducted for longer reports/smaller font. You will be marked on scientific content as well as
use of correct spelling, grammar, formatting and clarity. Remember - someone has to read
this!
I. Background/Introduction: Shows fundamental understanding of why we did the
experiment, how the experiment works, and what the advantages/disadvantages
are.
∙ Purpose of the experiment.
∙ Important background and/or theory (protein and methods).
∙ Description of specialised equipment.
∙ Justification of experiment's importance.
II. Methods
∙ Briefly describe procedure and any modifications to procedure (don't rewrite lab
script).
III. Experimental Results
∙ Present data in tables and graphs (don't forget to label all axes, number figures,
and provide titles).
∙ Refer to all tables and graphs in the text - i.e. use complete sentences to draw
attention to key points in tables or graphs.
∙ Provide a sample calculation for each type of calculation. This is the only way to
get partial credit for incorrect calculations. Also, for each calculation highlight the
final answer in bold so I don't have to hunt around (and possibly not find) your
answer.
∙ State key results in sentence form, and summarize results in tables.
∙ All figures must contain figure captions.
IV. Discussion
∙ This is the most important part of the report, where you can show your
understanding of the results of the experiments. Discuss the significance or meaning
of the results.
∙ Analyse and interpret results and analyse experimental error.
∙ Answer questions posed in lab.
V. Conclusion
∙ Very brief - did the experiment work and what did you learn?
VI. Appendices
1. Answers to questions from the lab script
2+. At your discretion
11
INTRODUCTION
__________________________________________________________________________
To complete the following experiments, you will be provided with a sample of your assigned
protein (same one as in data base workshop) of unknown concentration (U, approximately 1
mg/mL  30% ) and a sample of a protein standard solution of accurately known
concentration (S, exactly 1.0 mg/mL). Both samples will be dissolved in a buffer solution (50
mM sodium phosphate, pH 7.0) and you will be provided with additional buffer which you
should use to prepare all subsequent dilutions of both U and S.
*Important: You will be provided with > 1.5 times the volume of sample required to
complete all measurements in the labs. Be careful with it – you will not get more. You
should read through the entire lab script and calculate the amounts of these substances you
will need before starting. I also suggest you throw nothing away until you have completed
all the labs.
Use the table below to carefully calculate the amount required for each experiment. Get
this checked by a demonstrator in order to obtain your protein.
Experiment
I
Assay
A
B
C
D
Vol ~ 1 mg/mL U
Vol 1 mg/mL S
II
III
Total Volume
QUESTIONS (ANSWERS TO BE SUBMITTED IN APPENDIX 1 OF LAB REPORT)
___________________________________________________________________________
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?
12
EXPERIMENT I:
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 four different methods and evaluate their relative merits.
Solutions.
At this point, you will have two protein stock solutions which you will use throughout
Experiment I. The first is a stock solution of your protein of interest with an approximate
concentration of 1 mg/mL ( 30%), referred to here as protein stock U. The second protein
stock solution is that of a protein standard stock (denoted S), whose concentration is
accurately known and given on the bottle (exactly 1 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 the protein
of known concentration (made from S). Plot absorbance verses µg of protein in the assay
mixture. You may plot the data electronically or on graph paper, but one of these plots 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 lab 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.
ASSAY A: ABSORBANCE AT 280 NM.
___________________________________________________________________________
In this assay you will measure the A280 of the approximately 1 mg/mL U protein sample in
quartz cuvettes using a Perkin Elmer Lambda 25 spectrometer. You will then use this
information, along with the extinction coefficient determined in the data base exercise, to
calculate concentration.
13
Protocol.
To collect a baseline, put ~ 2 mL 18.2 M water in either a clean dry or clean water rinsed
quartz absorbance cuvette. Set the instrument parameters as follows:
Scan
Data interval
Start wavelength
Stop wavlength
Instrument
Scan speed
Lamp UV
Lamp VIS
Lamp change
0.1 nm
400 nm
200 nm
240nm/min
On
On
326 nm
Sample
Number of samples 1
Run a baseline/background spectrum with the cuvette in the sample position.
To obtain the sample spectrum, 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 3 times). Dry cuvette either with nitrogen line (or air if nitrogen is not
available). Pipette directly into the cuvette: 1 mL of your U; add 1 mL 18.2 M water.
Measure a spectrum using the same parameters as the baseline. Save your data directly
onto a memory stick as an ASCII file. Recover sample from the cuvette and keep it – you
may need it if you make a mistake later on.
QUESTIONS (ANSWERS TO BE SUBMITTED IN APPENDIX 1 OF LAB REPORT)
___________________________________________________________________
1. 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 U.
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).
2. 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.
14
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 S standards for a calibration curve containing: 0.0 mg protein;
0.25 mg protein; 0.5 mg protein; 1.0 mg protein; and 1.5 mg protein from the 1 mg / mL S
protein solution provided. Also prepare duplicates of 2 different concentrations of U that
you predict will lie within the range defined by your calibration curve (U 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 S
15
and use the curve to determine the concentration of U. Include this plot in your lab report,
and plot the data for your U solution on the curve as well. Finally, back calculate the
concentration of your original (~ 1 mg/ mL) stock.
* Important note: the Beer-Lambert 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.
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 dye 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.)
You will be using the Biorad assay, which is a commercial version of the Bradford protein
assay. The accuracy of this assay depends on accurate pipetting and above all thorough
mixing. You will be using automatic pipettes make sure that you know how to use them
before attempting the assay. There is a small error introduced into this method by the
calibration points having slight differences in volume. This error is small and can be ignored.
Protocol.
You will again begin by preparing S standards for a calibration curve containing: 0 µg, 5 µg,
10 µg, 20 µg, 50 µg, 100 µg of protein from the 1 mg / mL S protein solution provided. Also
16
prepare aliquots of two different concentrations of U that you predict will lie within the
range defined by your calibration curve (U should always be measured in duplicate). Place
required volumes, x µL, of the S and U solutions in clean, dry test tubes. Add 3 mL of the
diluted Biorad reagent and mix thoroughly (could vortex (avoid excess foaming) or mix
several times by gentle inversion of test tube). After a period of 10 minutes, determinine
A595. Plot A595 versus the amount of protein in each S sample. Include this plot of your
calibration curve in your lab report, and plot the data for your U solution on the curve as
well. Read unknowns U from the standard curve, and determine their concentrations.
Then back calculate the concentration of your original (~ 1 mg/ mL) stock, taking all dilution
into account.
ASSAY D: BCA METHOD
___________________________________________________________________________
Once again, the S solution will be used to prepare samples for a calibration curve. Table 3
below summarises the S 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. Make the U samples in duplicate, so you have two measurements of each
concentration.
Table 3:
Protein concentration
(g/mL)
1000
500
200
50
0
Vol. 1 mg/mL SS solution
(L)
100
50
20
5
0
Vol. buffer or water (L)
0
50
80
95
100
Protocol.
A stock reaction mixture containing 20 mL BCA reagent, Pierce No. 23223 and 285 L 4%
CuSO4 has been prepared for you. For each analysis: make 100 L protein solution of the
desired concentration in buffer or water. Mix well. Then add 2 mL of the reaction mixture.
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 S as a function of protein concentration. Include this plot of your calibration curve
in your lab report, and plot the data for your U solution on the curve as well. Read
unknowns U from the standard curve, and determine their concentrations. Then back
calculate the concentration of your original U (~ 1 mg/ mL) stock, taking all dilution into
account.
Prepare a table containing the concentrations of your US stock solution as determined by
Assays A, B, C, and D. Include this table in your lab report in the results section, and discuss
reasons for any differences.
17
EXPERIMENT II:
DETERMINATION OF PROTEIN MOLECULAR MASS BY SDS-PAGE
Aims. In order to determine the mass, purity and oligomeric states of your protein of interest, SDSPAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) can be used. SDS-PAGE is
a technique that is used to separate and identify proteins based on their size, which
correlates to their molecular weight. In this experiment, you will prepare and SDS-PAGE gel,
and you will run your U and S samples alongside a molecular weight standard.
Procedure for preparing an SDS-PAGE gel.
Begin by ensuring that you have one short glass plate and one taller, thicker glass
plate (this is the spacer plate), and that both are clean and dry. Be VERY CAREFUL with these
as they are quite fragile and break easily, and we don’t have many extras. Place the green
casting frame upright in front of you, with the pressure cams in the open position and facing
forward (see Fig.1).
Figure 1: Assembling the glass plates and casting frame.
Place the short glass plate on top of the longer plate so that you can see that there is gap in
between them – this is where the gel will be formed. Orient the spacer plate so that the
labelling is “up”, and then slide the two glass plates into the casting frame. Ensure that both
plates are flush on a level surface and that the label on the spacer plate is oriented
correctly. When the glass plates are in place, engage the pressure cams (rotate towards the
outside of the frame) to secure the glass cassette “sandwich” in the casting frame.
Now place the clear casting stand in front of you and place a gray casting stand gasket
(rubber pad) at the base of one of the two positions in the stand (Fig. 2). Gently place your
18
casting frame containing your glass slides on top of the gasket, ensuring that the bases of
both plates are flush with the gasket and the pressure cams are facing outward.
Figure 2: Placing casting frame and glass slide assembly onto the casting stand, and testing for leaks.
The gasket will prevent leaking once you begin pouring the gel. Finally, secure the frame
into the stand using the clips at the top. To check for leaks, fill the gap between the two
plates using with 1mL of water using a pipette (Fig. 2). If no leaking is seen, absorb the water
gently using a piece of filter paper. If you do see leaks, you will need to dry everything
carefully, and reassemble from the beginning.
Next you will make the 12% Tris-glycine resolving gel. Using the pipettes and tips provided,
and the pre-made solutions on the bench, begin by adding each of the following to a ~15 mL
capacity vial (in this order):





2.4 mL of deionized water
3.0 mL of 30% acrylamide mix
2.0 mL of 1.5 M Tris buffer (pH 8.8)
75 µL of 10% SDS
75 µL of 10% ammonium persulfate
The final component of this mixture starts the polymerization reaction, so once this is added
to the above solution, you’ll need to work quickly! When you’re ready, add

5 µL of TEMED to the solution and swirl gently to mix. Then, using a P-1000 pipette
and tip, draw up 1mL at a time and gently pipette the solution between the glass
plates. Repeat until you have used ALMOST all of the solution, but leave a small
amount of the mix in the bottom of the beaker. You will use this to gauge when your
polymerization is complete (it is difficult to tell otherwise).
Once your resolving gel has been pipetted between the glass plates, immediately cover it
with a thin layer of isopropanol
and then get started making the 5% stacking gel, which
19
will sit on top of the resolving gel. In a similar manner to before, you will use the pipettes
and pre-made solutions on the bench to prepare a solution in a vial containing (combine in
this order):





0.68 mL of deionized water
0.17 mL of 30% acrylamide mix
0.13 mL of 1.0 M Tris buffer (pH 6.8)
10 µL of 10% SDS
10 µL of 10% ammonium persulfate
Then, when you’re ready, add:

1 µL of TEMED
to the solution and swirl gently to mix. Working quickly,
gently pipette this solution (retaining a small amount to gauge when the
polymerization is complete) between the glass plates on top of the resolving gel, and
then insert a green comb carefully into the top of the gel. This will displace some of
the liquid, so be careful not to touch any of the liquid with your bare hands and
always wear gloves. Once the gel has set, mark the bottom of each well with a
marker.
Procedure for preparing protein samples and molecular weight standard
Obtain five empty 1.5 mL Eppendorf tubes and place in the rack at your bench. Using a
marker, label these tubes U 1, S 1, U 0.1, S 0.1, and MW (for MW marker). Using the P20
pipette, pipette 10 µL of your ~ 1 mg/mL U and 1.0 mg/mL S into the tubes labelled U 1 and
S 1, respectively. To each, add 10 µL of the 2X sample loading buffer (on the benchtop) and
mix gently by tapping the bottom of the tube. Prepare a 1 in 10 dilution of the U 1 and S 1
samples in the 2X sample loading buffer (U 0.1, S 0.1). To the tube labelled MW, pipette 5µL
of the MW marker (which already contains the sample loading buffer).
Procedure for loading and running the gel
A scheme for setting up the gel tank is illustrated in Fig. 3. As shown in Fig. 3A, set the
clamping frame on a clean flat surface with the clamps in the open position. Remove your
gel from the casting frame and place it onto the gel supports (moulded into the bottom of
the clamping frame) in the clamping frame, with the short plate facing inward. Your gel will
rest in the clamping frame at a 30° angle (Fig. 3B).
On the back of the clamping frame, place a buffer dam onto the supports which will also
rest at an angle. Then pull the gel and the dam towards each other so that they rest on the
green rubber gaskets in the clamping frame, making sure that the short plate in your gel sits
below the notch at the top of the green gasket. While holding your gel and the dam firmly
against the gaskets, slide the green arms of the frame over the gels to lock them into place
(Figs. 3C and 3D). Important: Do not try to lock the green arms of the frame without making
sure that the gel and dam are lined up perfectly in the module. As a demonstrator if you are
20
concerned. Once locked in place, put the entire clamping frame into the gel tank (Fig. 3E),
making sure that the red (+) electrode jack matches the red marking on the top right inside
edge of the tank and that the tank is resting flat on the benchtop.
Figure 3: Assembling the clamping frame and gel tank for SDS-PAGE. Modified from Figure 4 of the
Mini-Protein Tetra cell Instruction Manual, Bio-Rad.
Now dilute 50 mL of 10X running buffer on the benchtop with 450 mL of water to
make 1X running buffer, and pour a small amount of this into the space between the gel and
the dam in the clamping frame. If no liquid leaks out into the gel tank, you are ready to load
your samples. Fill the clamping frame the rest of the way up with 1X running buffer, then
slowly and carefully remove the comb from your gel. You should still see the marks that will
help you locate each well. Rinse the wells of the gel by gently pipetting running buffer up
and down each one. You will need to load the following samples into the wells:
Gel Lane
1
2
3
4
5
Sample
MW Marker
U1
U0.1
S1
S0.1
Volume to load
5 µL
10 µL
10 µL
10 µL
10 µL
When you are ready to load the gel, get a demonstrator to show you how to load the
first well. Then load the remaining samples using a P10 or P20 pipette and yellow tips into
21
gel lanes 1-5. Load the wells slowly, and allow the samples to settle to the bottom of the
wells. In the remaining lanes, load 10µL of 1X sample loading buffer (made by diluting 2X
sample loading buffer with water). Once all the lanes are loaded, fill the outside of the tank
with the remaining running buffer and place the lid on the tank, again making sure to align
the color coded banana plugs and jacks. Then insert the electrical leads into a suitable
power supply with the proper polarity. Set the power on the power supply to a constant
200V, and start running the gel. Run the gel for 35 minutes, at which point you should turn
off the power supply and disconnect the leads. Remove the tank lid and carefully lift out the
clamping frame, pouring the buffer down the sink. Carefully remove the gel from the
clamping frame, and finally release the gel by gently separating it from the glass slides. Place
the gel in a small container for staining. Rinse the gel tank, clamping frame and buffer dam
with water and leave to dry on paper towels on the benchtop.
Procedure for staining protein bands on gel
* Important note about Coomassie brilliant blue R-250: This is a very intense stain and
difficult to remove. To avoid ruining the new lab, as well as your clothes: (i) always wear
gloves; (ii) all work with the stain should be in the trays provided in the fume cupboard, as
the ceramic surface of the fume cupboard is resistant to staining, but make sure there is no
spillage on the lip of the fume cupboard as this is easily stained; (iii) if there is spillage, mop
up IMMEDIATLEY with the destaining solution or methanol.
Obtain 100 mL of Coomassie stain (1 g Coomassie brilliant blue R-250 dissolved in 1 L of 5%
glacial acetic acid
, 50% methanol
, and 45% deionized H2O) and pour into a small
container holding the gel. Stain the gel by rocking gently for 30 min – 1 hr on a rotating
platform. Longer staining helps resolve weak (less concentrated) protein bands, but the
longer the gel is exposed to stain, the longer it will take to destain. Pour the used stain into
the waste container provided (do not pour stain down the sink), then rinse the gel 2-3
times with a small amount of cold water to remove excess stain.
Obtain 100mL of the destaining solution (10% glacial acetic acid, 20% methanol, 70%
deionized water), and add enough of this solution to cover the gel in its container. Destain
the gel by rocking gently for 30 min– 1 hr on a rotating platform. For more rapid destaining,
you can add a small, bunched up tissue to the tray to absorb the Coomassie R-250 dye as it
is extracted from the gel. Pour the used destain solution into the waste container provided
(do not pour destain down the sink!). All gels should be disposed of in the waste solids
containers. When you have finished make sure all your glassware has been washed, dried
and put away.
Identification of proteins from migration on SDS-PAGE gel
You need to PHOTOGRAPH YOUR GEL as you will need to include a clearly labelled image of
this gel in your lab writeup – please bring a USB stick to the laboratory with you. Label the
lanes as well as all bands in the gel (including the MW marker masses). Discuss results in
terms of expected mass purity, and oligomeric state.
22
EXPERIMENT III:
PROTEIN SECONDARY STRUCTURE DETERMINATION BY CIRCULAR DICHROISM
Aims. To determine whether a protein is correctly folded under a specific set of solution
conditions, circular dichroism spectroscopy can be used to determine the percentage helix
and sheet. In this experiment, you will acquire a CD spectrum of your protein if interest, fit
the data using specialized software to estimate helix and sheet content, and compare this
with the secondary structure content as seen in the crystal structure.
Protocol.
The practical part of this experiment is straight forward and requires 200 L of 1 mg/mL
solution of your U protein to be put into a 1 mm quartz 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. Dependinng
on the quality of the spectrum obtained, you may need to repeat the experiment with 200
L of a 0.1 mg/mL U solution. Prepare this solution by dilution of 20 L of your 1 mg/mL U
solution.
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). Dry it with air or a
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 I. Determine the -helical content
of your protein as given below and compare the answer with that from the crystallographic
data base. Include the CD spectrum and the fit, as well as all calculations, in the results
section of your lab report.
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 subtracted 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.
Ensure 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
23
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