___________________________________________________________________________

MOAC DOCTOR AL TRAI NI NG CENTRENOVEMB ER/DECEMB ER 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___________________________________________________________________________

PAGEDeadlines and other important information 3Timetable 4Essay assignment 5Laboratory and workshop manual:

Workshop 1 6-8Protein List 8Workshop II 9Aims and Assessment 10Guidelines for preparation of lab report 11Experiment I 12-17Experiment II 18-22Experiment III 23-24

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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: Due by 5 pm on the day of the workshop.

Essay: Due Tuesday 19 November at 4 pm. NMR assessment: Due Tuesday 3 December at 4 pm. Laboratory reports: 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.

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BREAKDOWN OF MARKS___________________________________________________________________________ Assessed work: 45% Exam: 45% Attendance: 5% Laboratory conduct: 5%

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. 9:00-11:00: UV lecture and workshop (Rodger, MOAC Lecture Room)Nov. 5 11:00-13:00: Fluorescence lecture, & workshop, (DNA melting curve) (Rodger,

MOAC Lecture Room)14:00-17:00: Laboratory (Dixon, Chemistry B309)

WEEK 7: Mon. 9:00-11:00: CD lecture (Rodger, MOAC Lecture Room)Nov. 11 11:00-12:00: CD workshop (Rodger, MOAC Lecture Room)

14:00-16:00: Linear Dichroism lecture (Rodger, MOAC Lecture Room)

Tues. 9:00-10:00: Pre-lab (Dixon, MOAC Lecture Room)Nov. 12 10:00-17:00: Laboratory (Dixon, Chemistry B309)

WEEK 8: Mon. 9:00-12:00: Introduction to Mass Spectrometry (O'Connor, MOAC Lecture Nov. 18 Room)

Tues. 9:00-13:00: Introduction to NMR (Brown, MOAC Lecture Room)Nov. 19 14:00-17:00: NMR Group Work (Brown, MOAC Lecture Room)

*Deadline 1: Essay due (4 pm)

WEEK 9: Mon. 9:00-12:00: Bio-applications of high field NMR (Blindauer, MOAC Lecture Room)Nov. 25 13:00-16:00: X-Ray crystallography lecture and demo (Cameron, BSR5, Bio. Sci)

Tues. NMR Demonstrations Nov. 26 9:30-12:30 (MOAC Students): (Lewandowski, Milburn House)

10:00-12:00 (As:MIT Students): (Prokes, Milburn House)

WEEK 10: Mon. 9:00-17:00: EXAM Dec. 2

Tues. 10:00-11:00: Pre-lab (Dixon, MOAC Lecture Room)Dec. 3 11:00-16:00: Laboratory: (Dixon, Chemistry B309)

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*Deadline 2: NMR Assessment due (4 pm)

WEEK 10+1: Tues. Dec. 10 *Deadline 3: Lab reports & Feedback forms due

ESSAY___________________________________________________________________________Select one of the following essay topics:

1. The structures of membrane proteins2. Sugar-binding proteins: Structure and Function3. 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.

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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.

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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 “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___________________________________________________________________________

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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)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)

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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: __________________________________________________

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* Practice data set (monoclonal antibody fragment) available on CH921 website:

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.

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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. Appendices1. Answers to questions from the lab script2+. At your discretion

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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 Assay Vol ~ 1 mg/mL U Vol 1 mg/mL SI A

BCD

IIIII

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?

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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.

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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 0.1 nmStart wavelength 400 nmStop wavlength 200 nm InstrumentScan speed 240nm/minLamp UV OnLamp VIS OnLamp change 326 nm SampleNumber 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 mol1dm3cm1; Tyr, max ~ 1280 mol1dm3cm1; 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 mol1dm3cm1. The values for chymotrysinogen are: 25670, 8, 4, 10, 51340. Comment.

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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.

C

O

C H

N N C

H R H O

C u ( I I )

C

O

C H NNC

HRHO

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 37C. 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 37C. 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

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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

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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)Vol. 1 mg/mL SS solution

(L)Vol. buffer or water (L)

1000 100 0500 50 50200 20 8050 5 950 0 100

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 37C 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.

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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, SDS-PAGE (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

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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

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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 standardObtain 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 gelA 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 concerned. Once locked in place, put the entire clamping frame into the gel tank (Fig. 3E),

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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 Sample Volume to load1 MW Marker 5 µL2 U1 10 µL3 U0.1 10 µL4 S1 10 µL5 S0.1 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 gel lanes 1-5. Load the wells slowly, and allow the samples to settle to the bottom of the

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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.

EXPERIMENT III:

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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 by opening a DOS window within windows. Type ‘c:’. Then type ‘cd\cdsstr’ at the command

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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.

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