Research Paper Writing Help

PROTEIN PURIFICATION – A COMPUTER SIMULATION

A week ago, you read a paper in which various separation techniques were used.  This week, you will experiment with a computer program that simulates additional methods of separation.

Protein Purification was written by Andrew Booth of the University of Leeds as part of the eLABorate  project.  The project was funded under the Teaching and Learning Technology Programme by the four higher education funding bodies, HEFCE, HEFCW, SHEFC, and DENI.  © The University of Leeds, 1997.

There are two parts to this exercise. The first (Part I) is to be done before you arrive in lab through the following link:

http://www.agbooth.com/pp_ajax/

The protein purification in Part II is to be completed in lab, working individually.

To carry out this exercise, you must familiarize yourself with the program and the possible purification steps. Before coming to laboratory, complete Part I and review the “Written Work for Protein Purification Lab” found at the beginning of Part II.

Protein Purification

Aims

The aims of this computer simulation are:

to familiarize you with a range of protein separation techniques

to allow you to explore how these techniques work, and to see their constraints

to allow you to devise schemes to purify proteins from a mixture, using combinations of      techniques

How to use the program

Protein Purification is a Windows based program. It uses the standard menu bars and drop-down menus; these can be operated using the mouse or keyboard commands. This document assumes that you are reasonably familiar with Windows. If you have not used Windows before, ask a demonstrator for some help before you start. Throughout this document you will be given the necessary information to use each part of the program. If you follow the exercises in the order in which they are set, you will learn how to use the program as you progress.

Protein Purification has an extensive Help system including: more information about the various protein separation techniques; a list of the time costs of each of the techniques; clues about strategies that you might use; and a progress report of your current separation scheme. The Help menu is available from all parts of the program and you will also find Info buttons; press these to get instant help related to the task in hand.

When you want to finish using Protein Purification, choose Go home from the Quit menu. When you are asked if you want to store your material, answer No. (Remember that you will lose your work on the protein if you answer No, so choose Cancel to return to the program if you change your mind).

Tasks to perform using the program

The exercises are divided into two parts. Part I familiarises you with the program and allows you to explore the various protein separation techniques using a simple mixture of proteins. Once you are familiar with all of these methods, in Part II you will be asked to use combinations of separation techniques to produce a pure sample of a protein from a much more complex mixture.

The tasks in Part I require short answers to be completed on this sheet, preferably before coming to lab. You are strongly advised to read all the questions in a particular section before you begin working on the computer; this should save you time, as many of the questions may be answered from a single, carefully planned experiment. When you have completed Part I there will be a group discussion when you may be asked to present your answers to the rest of the class.

The report on your work in Part II will be completed in lab and handed in. You are strongly advised to follow the guidelines on presentation.

For Part I, complete the sections on (1) SDS-PAGE, (2) Ion-exchange chromatography, and (3) Gel filtration. Sections 5 – 7 on heat treatment, ammonium sulfate fractionation, and hydrophobic interaction chromatography may also be useful for your protein purifications in Part II, but they do not work with the protein mixture Easy3_Mixture (they do work for the more complicated mixture that you’ll be using in Part II). Isoelectric focusing (4) and affinity chromatography (8) also work, but you are not allowed to use them in your purifications!

PART I                     Protein Purification Techniques

Getting started

1. On the title page, click on Start and select Choose a Mixture (or Start from beginning) from the drop-down menu.

2. For the exercises in Part I choose the Easy3_Mixture.

3. Another dialog box pops up; this allows you to select the protein you wish to purify from the mixture by its number. For the first task choose protein 1.

4. A box appears with information about protein 1; when you have read it and noted the relevant information click OK.

5. Throughout the exercises in Part 1 you will need to keep returning to this menu to select another of the proteins from this simple mixture. To do this, from the Quit menu choose Abandon scheme and start again; then start again from the beginning.

It is a good idea before you begin the exercises, to explore the menus and to familiarise yourself with the program in general.

1)  SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis)

Introduction:SDS-PAGE is a separation technique used largely for analysis. It can be utilised to discover properties of the proteins that we wish to purify using other, more preparative, techniques. It also allows us to follow the progress of our protein purification. In preparing the protein sample for SDS-PAGE the proteins are denatured and SDS (which is negatively charged) is bound to the proteins in a constant mass ratio. The result is that all the proteins have almost a constant charge to mass ratio, and during electrophoresis will be separated solely on the basis of their size. An electric field is created across the polyacrylamide gel, and the negatively charged proteins migrate towards the anode (positive electrode). As they move through the gel, the larger molecules are retarded whereas the smaller molecules can pass more easily through the pores in the gel. The result of this sieving is that the smallest molecules would reach the anode first, and the largest last. However, the electric field is turned off before the proteins reach the end of the gel, and the mass of the proteins can be estimated from the distance travelled through the gel. To estimate the relative molecular mass (Mr) of the sample proteins, standard proteins of known Mr are run on the same gel to provide a reference. To reveal the proteins on the gel a stain, such as Coomassie blue, can be used. If the protein we are interested in has been isolated previously, a specific antibody may be available for it. If this is the case then the protein in question can be identified from the array of proteins in the mixture, using an immunoblot. Note that as the proteins are denatured prior to separation by SDS-PAGE, the Mr that can be determined is that of the individual subunits, and not of the native protein. If the protein only has one subunit then of course its Mr can be estimated directly using this technique. If a protein is composed of more than one type of subunit, then more than one spot can be seen for this protein on the gel.

It is often useful to be able to perform a 2-dimensional separation of the protein mixture. The first separation involves isoelectric focusing (IEF). IEF separates proteins on the basis of their pI (isoionic point), each protein migrates in an electric field through a pH gradient until it reaches a position where the pH of the surrounding buffer is equal to the pI of the protein (see section 4, p63 for more details). The IEF is performed in a rod gel (with no SDS present). Once the separation has been completed the rod gel is moulded to the top of a polyacrylamide slab gel containing SDS. As only small amounts of protein are involved, the SDS in the gel is sufficient to bind to the proteins as they migrate into the slab gel from the rod. Thus the second dimension is SDS-PAGE, with the proteins being separated by size.

What to do:From the PAGE menu, select 1-Dimensional PAGE.

Beware, once opened, never close the electrophoresis window from the control menu box (the square in the top left-hand corner of the window), otherwise you will not be able to perform any more electrophoresis. When you have finished a separation, click on Hide gel.

a) Perform an electrophoretic separation of the protein mixture using 1D SDS-PAGE.

(i) How many proteins appear to be present in the mixture? …………………………………………….(1 point)

(ii) Using the protein standards for reference, estimate the Mr of the subunits of each of the proteins in the mixture.

………………………………………………………………………………………………………………………………..(1 point)

(iii) Using the immunoblot (under PAGE select Immunoblot) identify which of the bands is protein 1.

………………………………………………………………………………………………………………………………..(1 point)

b) Now use 2D SDS-PAGE to analyse the simple protein mixture. (Choose Hide gel to leave the 1D SDS-PAGE screen; then select 2-Dimensional PAGE from the PAGE menu.)

(i) How many proteins appear to be present in the mixture now? ……………………………………..(1 point)

(ii) Explain what you would see if you only performed isoelectric focusing on this mixture.

………………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………(2 points)

(iii) What is the advantage of using 2D SDS-PAGE over the 1-dimensional technique?

………………………………………………………………………………………………………………………………………………..

…………………………………………………………………………………………………………………………………..(1 point)

c) Use the immunoblot facility to identify each of the proteins in the mixture, and complete the table. (This will involve selecting the proteins from this simple mixture in turn – see #5 on the previous page)

(3 points)

Proteinestimated Mrof subunit (kD)estimated pI
1  
2  
3  

d) In some of the exercises which follow, you will need to use 2D SDS-PAGE to discover the effectiveness of various separation techniques. You will therefore probably find it helpful sketch the positions of the proteins on this diagram, and label which one is which.

(3 points)

Text Box: 80-
60-
50-
40-
30-
Mr   20-
15-
10-
5-

Text Box: pH
3		4		5		6		7		8		9
|	|	|	|	|	|
e) (i) From the results of the 2D SDS-PAGE can you be certain that there are only three proteins present in this mixture?

………………………………………………………………………………………………………………………….(1 point)

(ii)Explain your answer to question e) (i).

………………………………………………………………………………………………………………………………………………….

………………………………………………………………………………………………………………………………………(1 point)

2)    Ion-exchange chromatography

Introduction: Ion-exchange chromatography separates proteins on the basis of charge using an ion-exchange resin. An ion-exchange resin consists of an insoluble matrix with charged groups covalently attached. A cation-exchange resin is negatively charged, and binds positively charged ions (cations). Similarly, a positively charged resin is called an anion-exchanger. An ion which binds weakly to the resin may be displaced, or exchanged, by an ion that binds more strongly. The degree to which a protein is retained by an ion-exchange column depends on the sign and magnitude of the protein’s net charge. The overall charge of a protein depends upon the number and type of ionisable amino acid side chain groups, and the pH of its surroundings. Each ionisable side chain group has a distinct pKa, that is the pH at which it is half dissociated. For each protein there will be a pH at which the overall number of negative charges equals the number of positive charges and so it has no net charge. This is its isoionic point (pI). If the pH is below the pI the protein, then the protein molecules are positively charged and will bind to a cation exchanger. If the pH is above the pI, then the protein is negatively charged, and so will bind to an anion exchange resin. A pH equal to the pI results in the protein molecule carrying no net charge and so it will not bind to either type of exchange resin. In selecting which type of exchange resin to use, it is important to consider the pH range over which the protein is stable (and therefore functionally active).

An ion-exchange resin is mixed with a suitable buffer, of an appropriate pH, to form a slurry. This is then poured into a chromatography column. The pH of the buffer will determine the charge on the proteins to be separated. The pH of the starting buffer should be at least one pH unit above or below the pI of the protein to be bound to the resin, to ensure adequate binding. It is also important to bear in mind the pH ranges of the ion-exchangers. A weak ion-exchanger is ionised over only a limited pH range (the term ‘weak’ does not refer to the strength of the binding of the ions to the resin). The resin in the column is washed with the starting buffer, then the protein mixture is applied. Proteins will bind or pass straight through the column, depending on their charge relative to that of the resin. Those that have been bound can be eluted by changing either the pH or the ionic strength of the eluting buffer. At low ionic strength there is minimal competition between the buffer ions and the proteins for charged groups on the ion-exchanger, and so the proteins bind strongly. As the ionic strength is increased the competition increases and so the interaction between the ion-exchanger and the proteins is reduced, causing the proteins to elute, regardless of the type of ion-exchanger used.

In this exercise you will investigate the binding of the proteins from the simple mixture, to two different ion-exchange resins, using both salt (ionic strength) and pH gradients.

What to do: Choose a pair of ion-exchange media to experiment with, either DEAE- and CM-cellulose, or Q- and S-Sepharose. (Tick the appropriate box).

ion-exchange mediumcharged groupmatrix typeI am using
DEAE-cellulose-CH2O-CH2CH2N+H(C2H5)2weak anion-exchanger 
CM-cellulose-CH2O-CH2COOweak cation-exchanger 
Q-Sepharosequaternary aminestrong anion-exchanger 
S-Sepharose-SO3cation-exchanger 

As this is the first time you will have encountered a chromatographic technique in this program, take some time to investigate the menus and the options available, using the following notes to help you. It is worth doing, as many of the other separation techniques in Protein Purification are presented in a similar way

Select Ion exchange chromatography from the Separation menu. A menu pops up; choose one of the ion exchange media from the top list by clicking on it (you can change your mind by clicking on another from the list) and an elution method from the bottom list; when you are happy with your selection click on OK. Another dialog box appears; type in the pH of the equilibration buffer and click on OK. A third box appears in which you enter the values for the start and end of your chosen type of gradient (either salt or pH), then click on OK. A graph, or “chromatogram” will appear. This shows the amount of protein, as detected by UV-absorption, (left-hand y-axis) against the fraction number (x-axis), and also the gradient used (scale on right-hand y-axis).

There are a number of things you can do from this screen, (and this is the same for the other separation techniques which produce a similar type of display).

ར  You can perform an enzyme assay on the fractions, to discover the location of the protein of interest. From the Fractions menu select Assay enzyme activity. A graph of enzyme activity is superimposed on the chromatogram.

ར  You can perform 1D SDS-PAGE on selected fractions. From the PAGE menu select 1-Dimensional PAGE. A box appears to tell you how to select up to 15 fractions for electrophoretic analysis. When you have read this, click on OK to continue, or cancel if you have changed your mind. This is another useful way of finding your selected protein, and checking for contaminants in a chosen fraction.

ར  You can perform 2D SDS-PAGE on selected fractions. From the PAGE menu select 2-Dimensional PAGE. Again a box appears to tell you how to indicate the fraction to be analysed.

ར  If you want to select a certain group of fractions for use in the next step of your purification scheme, from the Fractions menu choose Pool fractions. A box appears, telling you how to use the mouse to select the fractions; you can cancel at this stage, or click on OK to continue. Slide the arrow to the first of the fractions, click the left-hand mouse button, point the arrow to the last of the fractions and click with the mouse again. Select carefully with the mouse, as you do not get the chance to undo your selection. A results box appears; click on OK to continue. (The results in this box are summarised in Progress report, available from the Help menu.)

ར  If you are unhappy with the results of this separation step, you can choose to Abandon this step and continue (available in the Quit menu). (NB You do not have this option if you have already pooled your fractions.).

ར  If you wish to abandon your entire separation scheme, choose Abandon scheme and start again from the Quit menu. If this option is not available, choose Abandon this step first, and then Abandon scheme on the next screen. You will be asked if you really want to do this (as it means that you lose all your data), if you choose No you will be returned to the screen you just left, if you choose Yes, the box appears saying Start from stored material?

a) Using your anion exchanger with salt elution, perform three experiments using an increasing salt gradient of 0-1 M, one at pH 5, another at pH 7 and the third at pH 8.

(i) Report your results using the table (enter fraction numbers in the form 27-38, and indicate the proteins present in each peak by their identification numbers):

(6 points)

pHPeak 1Peak 2
 Fraction numbersProteins presentFraction numbersProteins present
5    
7    
8    

(ii) At pH 5, what is the charge (positive or negative) on protein 1?……………………….

Protein 2?………………………….   Protein 3?…………………………… (1 point)

What is the charge of the column (the anion exchanger)?………………………………………………….(1 point)

Will any of the proteins bind to the column? If so, which ones?…………………………………………(1 point)

At pH 7, what is the charge on protein 1?…………….. Protein 2?…………….. Protein 3?…………..  (1 point)

Will any of the proteins bind to the column, and if so, which?…………………………………………….(1 point)

At pH 8, what is the charge on protein 1?…………….. Protein 2?…………….. Protein 3?…………… (1 point)

Will any of the proteins bind to the column, and if so, which?…………………………………………….(1 point)

(iii) At pH 7, which peak (peak 1 or peak 2) represents proteins which bound to the column? ……………

Which peak represents proteins which did NOT bind to the column?………………………………….(1 point)

b) How would your results differ if you had used a cation exchanger? Be specific! (1 point per pH)

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

c) (i) Use the anion exchanger to investigate the effects of changing the pH gradient on the separation of the protein mixture and record the results in this table. (6 points)

pH of equilibration bufferpH gradient  Peak 1  Peak 2
  Fraction numbersProteins presentFraction numbersProteins present
55-8    
88-5    
77-5    
      

(ii) Comment on the results you observed. (3 points, one for each pH)

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

d) (i) Which protein can be separated in a single step using ion-exchange chromatography?

…………………… (1 point)

(ii) Explain your answer (1 point)

…………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………….

3)    Gel filtration

Introduction: Gel filtration separates proteins on the basis of differences in their size and shape. The technique uses a gel matrix consisting of porous beads of an inert, highly hydrated gel. The gel beads are packed into a glass or plastic column, and then equilibrated with a suitable buffer solution. The protein mixture is applied to the top of the column and then buffer is added to elute the proteins from the column. The eluate is collected at the base of the column as a series of fractions. As the proteins pass down the column they penetrate the pores of the gel beads to different extents, and so travel down the column at different rates. All proteins which exceed the maximum size of the pores will be unable to enter the beads. Therefore these proteins will only pass through the solution between the beads, and so elute from the column first, in the exclusion (or void) volume. All proteins smaller than the minimum size of the pores will equilibrate completely with the buffer inside and outside the gel beads, and so spend a proportion of their time inside the beads. These proteins will therefore move more slowly through the column and will be eluted last. These proteins elute in a volume very close to the bed (total) volume of the column. The pores in the beads are not all exactly identical in size, but span a narrow range of sizes. Proteins that have sizes very similar to the range of pore sizes will be excluded from some pores, whilst entering others. These proteins of intermediate size will therefore be partially excluded from the beads to an extent that depends on their size and shape. They will elute from the column in order of molecular mass, with the largest proteins eluting first and the smallest proteins last.

What to do: Use the various gel media available in this program to investigate the principles of gel filtration. There are three series of gel media to choose from, and each gel type is available with a range of pore sizes:

Gel-filtration media
Matrix namegel typeApproximate fractionation range for peptides and globular proteins (molecular mass)
Sephadex G-50adextran1500 -30 000
Sephadex G-100adextran4000 -150 000
Sephacryl S-200 HRadextran5000 -250 000
Ultrogel AcA 54bpolyacrylamide/agarose6000 -70 000
Ultrogel AcA 44bpolyacrylamide/agarose12 000 -130 000
Ultrogel AcA 34bpolyacrylamide/agarose20 000 -400 000
Bio-Gel P-60cpolyacrylamide3000 -60 000
Bio-Gel P-100cpolyacrylamide5 000 -100 000
Bio-Gel P-300cpolyacrylamide60 000 -400 000

aSephadex is a registered trademark of Pharmacia-PL

b Ultrogel is a registered trademark of Pharmacia-LKB

c Bio-Gel is a registered trademark of Bio-Rad Laboratories, Inc.

From the separation menu select Gel filtration. A menu pops up; choose one of the gel media from the list by clicking on it, you can change your mind by clicking on another from the list; when you are happy with your selection click on OK. A graph, or “chromatogram” will appear. This shows the amount of protein, as detected by UV-absorption, (y-axis) against the fraction number (x-axis).

a) Using the series of Ultrogel media examine the effect of increasing the pore size of the gel on the separation of the protein mixture.

(i) Complete this table with the fraction numbers occupied by each peak (eg. 32-45): 3 points

 Ultrogel AcA 54Ultrogel AcA 44Ultrogel AcA 34
1st peak   
2nd peak   

(ii) What conclusion can you make regarding the time of elution of a particular protein, as the pore size of the gel is increased? (2 points)

…………………………………………………………………………………………………………………………………………

(iii) Using this technique is it possible to separate the following pairs of proteins from each other? Complete the table below using yes if a pair can be separated and no if they cannot. (3 points)

protein 1 from 2protein 1 from 3protein 2 from 3
   

(iv) Reconcile the results observed here with those you recorded in the table for exercise 1) c).

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………..(2 points)

b) Perform a separation of the protein mixture using Sephadex G-50.

(i) In which fractions are the proteins eluting? ……………………………………………………… (1 point)

(ii) What do you think has happened to the proteins in the first peak?

…………………………………………………………………………………………………………………………(1 point)

(iii) Suggest how the resolution of this separation could be improved, using the Sephadex gel series.

……………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………(2 points)

c) Perform a separation of the protein mixture using Bio-Gel P-300.

(i) What results do you observe? …………………………………………………………………………..(1 point)

(ii) Explain what has happened to the proteins during this separation.

…………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………….(1 point)

(iii) Under what circumstances would Bio-Gel P-300 be the most suitable gel medium to separate a group of proteins?

………………………………………………………………………………………………………………………….(1 point)

The point values add up to 60 points; your score will be divided by 3 to give a possible total of 20 points.

4)    Isoelectric focusing(IEF)

Introduction:Isoelectric focusing (IEF) is a method for separating molecules which differ in their charge characteristics. For IEF of proteins, the protein mixture is subjected to an electric field in an inert support medium in which a stable pH gradient has previously been generated. The inert support can be either agarose or polyacrylamide. The pH gradient is formed in this by including a mixture of low molecular mass “carrier ampholytes”. The anode (positive electrode) region is at a lower pH than the cathode (negative electrode). The pH range is chosen such that the proteins to be separated have their isoelectric points within this range. A protein which is in a pH region below its pI will be positively charged, and so will migrate towards the cathode. However, as it migrates, so the pH that the protein experiences will decrease until the protein reaches a pH which is equal to its pI. At this point it has no net charge and so migration ceases. Should the protein overshoot this point, it will enter a region of pH above its pI and so become negatively charged. It will then reverse its direction of migration and now migrate towards the anode. Therefore proteins become focused into sharp stationary bands, with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by only a single charge being resolved. It is important to avoid molecular sieving effects so that the protein separation occurs solely on the basis of charge, so the chosen support medium has pores larger than the size of the proteins being separated. IEF is mainly an analytical tool, but can be used to prepare very small amounts of pure protein. In preparative IEF, if the separation has been performed in a slab gel or in a tray of gel beads, then the bands can be cut from the gel and the proteins eluted using a buffer solution.

What to do: Select Preparative isoelectric focusingfrom the Separation menu. A box appears in which you enter the pH values for the start and end of the pH gradient, then click on OK.

In this exercise you are going to explore the effects of pH range on the separation of proteins by isoelectric focusing. Conduct your own experiments and report briefly on your findings and conclusions . Use the following suggestions to help you:

1.    taking a wide range around the pI of the chosen protein

2.    using a narrow range around the pI of the chosen protein

3.    using a range which excludes the pI of the chosen protein

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………

5)    Heat treatment

Introduction: Protein purification procedures are usually carried out at low temperature (0-4C) since most proteins are stable at low temperatures. As the temperature increases from 0C to 37-40C their stability decreases significantly. Above 40C or so, most proteins become increasingly unstable and denature. At neutral pH, denatured proteins usually precipitate. Individual proteins differ in their heat sensitivity and so this can be used for purification purposes. The temperature stability of the desired protein is determined by trial experiment, for example by following enzyme activity as the protein mixture is incubated at different temperatures, for a set period of time. The minimum temperature at which gross inactivation occurs is noted. Once this temperature is known, less stable proteins can be preferentially inactivated by incubating the crude protein mixture at a temperature 5-10C below this value for 15 to 30 minutes. Since denaturation of all cell proteins occurs to some extent at all temperatures, and only increases with increasing temperature, the total activity of the desired enzyme usually falls to some extent after heat treatment. However, it may be a useful early step for the purification of rather more heat-stable proteins.

What to do: Use Heat denaturation (available in the Separation menu) to answer these questions:

a) Which of the proteins in this simple mixture is suitable for purification using heat treatment?

………………

b) Explain your choice of protein for question a).

…………………………………………………………………………………………………………………………………………..

c) For how long does the protein mixture need to be held at the following temperatures before all of the contaminating proteins are precipitated?

(i) 60C ……………………………………..

(ii) 50C …………………………………….

d) Why, in practice would it not be a good idea to perform a one step protein purification in this way?

…………………………………………………………………………………………………………………………………………

6)    Ammonium sulphate fractionation

Introduction: The solubility of proteins varies according to the ionic strength, and hence according to the salt concentration, of the solution. Two distinct effects are observed. At low concentrations of salt, the solubility of the protein increases with salt concentration. This phenomenon is called “salting in”. However, as the salt concentration (ionic strength) is increased still further, the solubility of the protein begins to decrease. At sufficiently high ionic strength the protein will be almost completely precipitated from solution – an effect called “salting out”. Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. The commonly used salt is ammonium sulphate, as it is very water soluble and has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution). Before carrying out a bulk separation, a test is performed to discover the salt concentrations to use. In this preliminary test the concentration of ammonium sulphate is increased stepwise, and the precipitated protein is recovered at each stage. Each protein precipitate is dissolved individually in fresh buffer and assayed for both total protein content and the amount of the desired protein (in this case an enzyme, measured by its activity). The aim is to find an ammonium sulphate concentration which will precipitate the maximum proportion of undesired protein, whilst leaving most of the desired enzyme still in solution. The precipitated protein is then removed by centrifugation and then the ammonium sulphate concentration of the remaining solution is increased to a value that will precipitate most of the enzyme whilst leaving the maximum amount of protein contaminants still in solution. The precipitated enzyme of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification. This technique of ammonium sulphate fractionation is extremely useful to quickly remove large amounts of contaminant proteins, as a first step in many purification schemes. It is also often employed at later stages of purification; to concentrate protein from dilute solution following procedures such as gel filtration.

What to do: Use Ammonium sulphate fractionation (available in the Separation menu) to discover a suitable protocol for protein purification using this technique. In order to carry out preliminary tests on the protein mixture, in the first box type the chosen concentration of ammonium sulphate, click on OK, a results box appears. If you wish to go back and try another concentration click on cancel in this results box, but if you wish to continue to another step in the purification, choose whether you want to use the precipitate or the supernatant by clicking on the appropriate word, then click on OK.

a) What is the maximum concentration (to the nearest whole number) of ammonium sulphate that can be added to this protein mixture without precipitating any of protein 2?

………………………………………………………………….

b) What is the minimum concentration (to the nearest whole number) of ammonium sulphate that can be added to the protein mixture to precipitate all of protein 2?

………………………………………………………………….

c) Is it possible to obtain a pure sample of any of the proteins in this mixture using ammonium sulphate precipitation alone?

………………………………………………………………….

7)    Hydrophobic interaction chromatography

Introduction: This technique separates proteins on the basis of their binding to and elution from a hydrophobic matrix, usually octyl- or phenyl-agarose. Binding of the proteins is often carried out at a high salt concentration to favour hydrophobic interactions. Some proteins may precipitate at this high ionic strength and so need to be removed by centrifugation prior to loading the protein mixture onto the column. Selective elution of bound proteins is then carried out by applying a decreasing salt gradient.

What to do: Explore the effects of changing the starting and final salt concentrations on the separation of the three proteins using Hydrophobic interaction chromatography(select it from the Separation menu).Choose either Phenyl- or Octyl Sepharose (use the same one for the whole of this exercise) and click on OK. Enter the values the Start and End of the salt gradient in the boxes provided, and click on OK.

I am using: (check one)

Phenyl-Sepharose 
Octyl-Sepharose 

a) What are the two effects of increasing the initial salt concentration on the elution profile?

(i)………………………………………………………………………………………………………………………………………

(ii)……………………………………………………………………………………………………………………………………..

b) What are the effects of increasing the final salt concentration on the elution profile?

…………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………

c) Which protein can be separated in a single step using this technique, without losing any activity?

…………………………………………………………………………………………………………………………………………

d) Investigate a series of fractions through the second peak eluted using 1D-SDS PAGE, what do you notice?

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8)    Affinity chromatography

Introduction: Affinity chromatography relies on the preparation of a matrix to which the protein of interest, and preferably only this protein, will bind reversibly. The matrix is usually beaded agarose, polyacrylamide or cross-linked dextran, to which a ligand has been covalently attached. The chemical nature of the ligand is determined by the known biological specificity of the protein to be purified. In the case of an enzyme, the ligand chosen would probably be a substrate or a reversible inhibitor or activator. If it is not possible to use a ligand that is absolutely specific to the molecule of interest, it is often possible to use a group-specific ligand. The matrix bearing the ligand is packed into a column, in a buffer that will be optimal for the protein-ligand binding. For example, if the ligand is an enzyme substrate, then the buffer must contain any co-factors that are required for binding. The buffer usually has a fairly high ionic strength, to minimise non-specific binding of other proteins to the ligand. The sample is applied at the top of the column and washed through the matrix. Ideally, only the protein of interest should bind. It can then be eluted specifically by the addition of a relatively high concentration of substrate or competitive inhibitor, or, failing this, by changing the pH and/or the ionic strength to disrupt the enzyme-ligand interaction. An alternative protocol can be used if an antibody, specific to the protein of interest, is available. This procedure is applicable to all proteins irrespective of their functional activities. The antibody is covalently coupled to a suitable matrix filling the column. Only the required protein will bind to the antibody and can then be eluted by procedures which weaken the antibody-antigen interaction. Affinity chromatography is a potentially powerful technique, but it can only be used when the functional activity of the required protein is known and a suitable ligand is available, or when a suitable antibody to the protein has already been obtained. Unfortunately, in many cases neither condition is satisfied, and so other protein purification methods have to be relied upon.

What to do: Use Affinity chromatography (from the Separation menu) to discover suitable purification methods for the proteins in this mixture. You will need to experiment, as you have no way of knowing the affinity of the various monoclonal antibodies available for each protein. Select a ligand from the left hand list, and choose what you want to elute the proteins with, from the list on the right, then click on OK.

a) For protein 2, discover which monoclonal antibody and elution system gives the best separation and yield.

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b) For protein 1, explain the results you observe when using monoclonal antibody MC01C.

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c) For protein 3, compare the results you observe when using monoclonal antibody MC03C with those for the polyclonal IgG, and explain any differences you observe.

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d) (i) Which protein has a competitive inhibitor that can be immobilised? ……………………………………………………..

(ii) Which is the most efficient method for performing a separation using this competitive inhibitor? Explain.

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(iii) How does the yield of protein for the method in d) (ii) compare to the best yield available for this same protein using other methods of affinity chromatography?

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9)    And finally………..

Suggest a two step purification process capable of separating all three of the proteins from each other. There is no single correct answer to this, we will discuss the various options available in the group discussion.

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PART II.  Purifying a protein from a complex mixture

WRITTEN WORK FOR PROTEIN PURIFICATION LAB

Answers to the following questions are due before you do the computer exercise.  All but one can be answered by using the previous sections.

Questions:

1.  Since proteins are quite fragile (labile) molecules, all purification steps should take into consideration the following conditions

1.

2.

3.

2.  Early in purification, two low resolving steps often used are

1.

2.

while a low capacity but high resolution step often used late in purification

is______________________________.

3.  Traditionally, one unit of enzyme activity is defined as

4.  As the enzyme is purified from a mixture of proteins we measure its purity by determining the number of units of enzyme activity per milligram of protein, this measure is the term _____________ _____________of the enzyme.

5.  “Fold” purification compares the _____________________of a fraction to that of the original mix.

6.  Enzyme yield is defined as:

7.  We will consider our purification is probably complete when the apparently pure protein yields one spot when tested by _________________________.

8.  When choosing a gel type for optimal fractionation of complex proteins the gel pore size is such that the desired protein is _____________________________________________.

9.  The ph at which a protein has no net charge is its ________________________________.

At this point it (will, will not) bind to an ion exchange resin.  Below this pH it will assume a __________ charge and bind to a (cation, anion) exchanger.

10.  DEAE cellulose or CM cellulose are effective only in the pH range ___to____.  The starting buffer should be of reasonably __________ ionic strength the affinity of proteins for the ion exchange resins (increases, decreases) as ionic strength (increases, decreases).

11.  The O.D.280  is monitored in each of the eluted fractions because

__________________________________________________________________________

___________________________________________________________________________.

Turn in the “Record of Purification” for two enzymes as well as the 2-D Page gel with the enzymes of interest circled. Be sure to identify them by number.  Remember it is possible that one of your enzymes is a dimer or that the isoelectric point of your enzyme is beyond the range of pH values displayed on the gel.

The report should concisely summarise your results (yield, purity and so on) for each stage of the purification, and your conclusions. You should say why you have chosen to use particular separation techniques for a given protein. The report should also include any relevant details regarding the characterisation of the proteins in question, for example estimated Mr of the subunit. Your report should conclude with your recommendations for the way in which the project should proceed, in terms of the optimum separation technique for a given protein. You do not have to report everything you have done, but if you discover that a particular separation technique is entirely unsuitable for a given protein, you could mention this, to save time for other researchers in the future.

It is strongly recommended that you keep careful notes of everything you do while you are carrying out your investigations; remember that the program does not record everything for you. However, you should not include all that you write down in your final report. Your notes will form the equivalent of a lab note book; you would not publish the entire contents of such a book as a scientific paper!

You will be assigned2 proteins to purify; you must clearly write the number of the protein to be purified at the start of the relevant section of your report. Failure to do this will result in your assignment not being marked.

You will purifyeach of your 2 proteins from a complex protein mixture in a crude mucosal extract. In each case the protein you must purify has not been isolated before. You are aware of its enzymic activity, so you can detect its presence, but there are no specific antibodies available. You have to discover the most efficient and cost effective separation method for each of the proteins assigned to you, so you need to carry out several investigations using different combinations of separation techniques to discover the optimal method. You are aiming to get a pure sample of your protein, with a high yield. The budget allocated to your project is restricted, so you must pay careful attention to the cost of each step in the purification. Your research director will monitor the progress of your project, both in terms of time and financial costs, and will intervene if it appears that you cannot meet the time and budget targets.

The proteins I have been assigned to purify from the crude extract are:

…………………………&…………………………..

What to do: BeginProtein Purification. On the title page, click on Start and select Choose a mixture from the drop-down menu. Choose the Default Mixture and type in the number of your assigned protein.

A common strategy is to run a separation, pool your samples, and then run a 2D electrophoresis experiment to evaluate your results.  A quirk of this program is that you then have to select “hide gel” from the menu before you can continue.

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