KE0026 Biochemistry Labs

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Wet lab: Protein purification and analysis

The purpose of this practical is to demonstrate different types of column chromatography that are used in the purification of proteins. We will also show how to do an analysis of protein fractions with SDS-polyacrylamide gel electrophoresis.

1. Determination of native molecular weight of proteins by gel filtration.
2. Desalting of a protein solution by gel filtration.
3. Packing of and separation on an ion exchange column.
4. Affinity chromatography: Binding of trypsin to a Benzamidine Sepharose column.
5. Separation of proteins on an SDS-polyacrylamide gel and determination of their subunit molecular weights.

Introduction
An example of a typical purification scheme is given in the figure below. This particular sequence of steps is of course not applicable in all cases which often means that a unique purification protocol has to be developed for each new substance you wish to isolate. Most important is that the steps complement each other and that the degree of purity increases each time. The number of steps in the protocol depends on the state of the starting fraction and on how pure you want your substance.

                  Crude cell extract
                          |
                  Streptomycin sulfate precipitation (removes nucleic acid)
                          |
                  Ammonium sulfate precipitation
                          |
                  Hydrophobic interaction chromatography (HIC)
                          |
                  Ion exchange chromatography
                          |
                  Gel filtration

To get a good recovery of the substance i.e. minimizing losses it is desirable that each step is as specific as possible. To check purity and yield you may use absorbance measurements, various types of electrophoresis and preferably also some kind of activity measurement.
The column chromatography part may be performed in different ways more or less manually. In the course practical we will use both manual ways as well as a system with pump and fraction collector. For more routine purifications this system can be built out to monitor absorbance etc. and quite often one uses a FPLC which is a programmable system with more powerful pumps.

Gel filtration
Gel filtration is used to separate proteins of different sizes. You may also determine the native molecular weight of a protein by this method since there is a linear correlation between the elution volume of proteins and the logarithms of their molecular weights (MW) (see experiment 1).
The system contains two phases, one stationary and one mobile. The stationary phase usually consists of a cross-linked polysaccharide which forms porous beads. The mobile phase normally consists of a buffer. The separation depends on the ability of molecules to enter the pores. Smaller molecules can diffuse into the beads and move more slowly down the column. Molecules are therefore eluted in order of decreasing molecular size. By varying the degree of cross-linking the gels are optimized for different molecular weight ranges.

Elution profiles
The result from a gel filtration experiment is often plotted as the variation of substances eluted as a function of the elution volume, Ve (see figure below). Ve is however not the only parameter needed to describe the behaviour of a substance since this also is determined by the total volume of the column and from how it was packed.
By analogy with other types of partition chromatography the elution of a solute may be characterized by a distribution coefficient (Kd). Kd is calculated for a given molecular type and represents the fraction of the stationary phase that is available for the substance. In practice Kd is difficult to determine and it is usually replaced by Kav since there is a constant relationship between Kav:Kd. Kav is obtained from

Kav = (Ve-V0)/(Vt-V0)

The total volume of the column (Vt) is simply calculated from p x r² x h and the void volume (V0) is determined by passing a large substance that does not interact with the beads (like blue dextran) through the column.

Ion exchange chromatography
The ability to reversibly bind molecules to immobilised charged groups is used in ion exchange chromatography (IEC). Which type of charged group one choses - positive or negative - depends on the net charge of the protein which in its turn depends on the pH. IEC is maybe the most commonly used technique today for the separation of macromolecules and is almost always included as one of the steps in the purification protocol. The experiment may be divided into four different parts:

1. Equilibration of the ion exhanger in a buffer in such a way that the molecule(s) of interest will bind in a desirable way.
2. Application of the sample. Solute molecules carrying the appropriate charge are bound reversibly to the gel. Unbound substances are washed out with the starting buffer.
3. Elution with a gradient of e.g. NaCl. This gradually increases the ionic strength and the molecules are eluted. The solute molecules are released from the column in the order of the strengths of their binding i.e. the weakly bound molecules elute first.
4. Substances that are very tightly bound are washed out with a concentrated salt solution and the column is regenerated to the starting conditions.

Affinity chromatography
This is a type of adsorbtion chromatography in which the component to be purified is specifically and reversibly bound to a ligand that has been immobilized on a matrix. Any component may be use as ligand as long as it can be covalently attached to the chromatographic bed material. Examples of this type of chromatography is antigen-antibody, enzyme-substrate analogue etc.

SDS-Polyacrylamide gel electrophoresis
Sodium Dodecyl Sulfate-PolyacrylAmide Gel Electrophoresis (SDS-PAGE) is an excellent and commonly used method to analyze purity and homogeneity of protein fractions. It may furthermore be used to estimate the molecular weight of protein subunits.
In general, fractionation by gel electrophoresis is based on differences in size, shape and net charge of macromolecules. Systems where you separate proteins under native conditions cannot distinguish between these effects and therefore proteins of different sizes may have the same mobility in native gels. In SDS-PAGE this problem is overcome by the introduction of an anionic detergent SDS which binds strongly to most proteins. When hot SDS is added to a protein all non-covalent bonds are disrupted and the proteins aquire a negative net charge. A concurrent treatment with a disulfide reducing agent such as b-mercapto ethanol or DTT (dithiothreitol) further breaks down the macromolecules into their subunits. The electrophoretic mobility of the molecules is now considered to be a function of their sizes i.e. the migration of the SDS-treated proteins towards the anode is inversely proportional to the logarithms of their molecular weights, or more simply expressed: Small proteins migrate faster through the gel. Compare this with the situation in gel filtration.
The polyacrylamide gel is formed by co-polymerization of acrylamide monomer CH₂=CH-CO-NH₂ and a cross-linking monomer N,N'- methylene bisacrylamide, CH₂=CH-CO-NH-CH₂-NH-CO-CH=CH₂ (bisacrylamide). To polymerize the gel a system consisting of ammonium persulfate (initiator) and tetramethylene ethylene diamin (TEMED) is added to generate a free radical. TEMED causes free radical generation from the ammonium persulfate which in its turn catalyzes the polymerization. The concentration of the monomers may be varied to give gels of different density. Usually gels with 10-15% acrylamide are used and the ratio bisacrylamide:acrylamide is 2.7-3.3%. In addition a so called stacking gel is cast on top of the separation gel. This has a lower concentration of acrylamide and lower pH in comparison with the main gel.

Experiment 1. Determination of column parameters by gelfiltration
A column packed with a Sephacryl gel will be used to separate macromolecules of different sizes. The native molecular weights of the proteins will be determined.

• Column (2.6 x 25 cm) filled with Sephacryl S-300. Total volume ~150 ml
• Separation equipment - peristaltic pump and fraction collector with 35-40 test tubes
• 500 ml of de-gassed 25 mM TrisHCl pH 8.0, 0.2 M NaCl
• Spectrophotometer (UV-lamp on)
• 3 % Hydrogen peroxide, H₂O₂
• Sample solution that contains the following molecules:

  Cytochrome c (horse heart)   2 mg/ml  MW=12.000, strongly red/brown
  Ovalbumin (hen egg white)    4 mg/ml  MW=43.000, colourless
  Catalase (bovine liver)      5 mg/ml  MW=240.000, weakly brownish
  Rubisco (spinach)            1 mg/ml  MW=550.000, colourless
  Blue dextran               0.5 mg/ml  MW=2.000.000, strongly blue
  

1. Equilibrate the column with 2-3 column volumes of degassed 25 mM Tris HCl pH 8.0 containing 0.2 M NaCl. Recommended flow rate is approximately 150 ml/h. Check this by putting a cylinder at the outlet of the column and measure the volume over 5 min. Switch on the fraction collector and fill it with 30-35 test tubes. From now on both pump and collector are controlled by the run/end button on the fraction collector. Use time mode for collection. Plug in the value of the time that is needed to collect 5-ml fractions. This usually corresponds to about 2 min depending on the exact flow rate. Check that the fraction collector works properly (changes fraction) by letting a few ml:s of buffer go though. Stop the pump again.
   
   
2. Apply the sample by putting the end of the tubing connected to the top of the column into the test tube with your protein sample and subsequently starting the pump. The total sample volume should not exceed 1-2% of the column volume. Stop the pump just before the sample is finished. Be careful not to get air bubbles in the system.
   
   
3. Switch back to the starting buffer and start the pump again. Elute the proteins at the flow rate mentioned above. Start the fraction collector which should be filled with test tubes and programmed in a suitable manner. Use time mode for collection. A recommended fraction volume is 5 ml which corresponds to 2-2.5 min depending on the exact flow rate. Observe the separation of the different molecules and how they move down the column. Measure the absorbance at 280 nm for each fraction. Some of the proteins are coloured which makes the evaluation more simple. The catalase may be detected with H₂O₂ which is cleaved to its products water and oxygen by this enzyme producing air bubbles. Note the elution volume Ve for each molecule. The elution volume for blue dextran serves as V0. Continue the washing until all proteins have eluted.

Data, results and discussion points that should be included in your lab report
1. Construct two graphs. One where you plot A280 as a function of fraction number. From this graph it should be possible to estimate Ve for each molecule. The second graph should be a plot of Kav against log MW of the proteins.
2. In which order are the molecules eluted? Why?
3. How can you use the second graph to determine the molecular weight of an unknown sample?
4. Why is the absorbance monitored at 280 nm. Could you use some other wavelength?
5. Which alternative methods can you think of to detect protein in the fractions?

Experiment 2. Desalting of catalase with a PD10 column
This type of column can be purchased ready to use. It is packed with a Sephadex G25 gel (gel filtration substance) and the total volume of the column is 9 ml. A coloured salt potassium dichromate is used to better demonstrate the desalting process.

• PD10 column with Sephadex G25
• 50 ml 25 mM TrisHCl pH 8.0
• Pipettes, tips and test tubes
• Catalase (10 mg/ml) and potassium dichromate (1 mg/ml)
  
• 3% Hydrogen peroxide H₂O₂

1. Mount a PD10 column vertically in a stand. Take off the lid and remove the liquid on top of the filter. Also remove the seal at the bottom of the column. Equilibrate the column with 20-25 ml of buffer.
   
   
2. Put 10 small test tubes in a rack and put the column above the first tube. Apply 1.0 ml of a solution containing catalase (10 mg/ml) and potassium dichromate (2 mg/ml).
   
   
3. Elute stepwise with 1.0 ml of the buffer and collect at the same time 1 ml fractions. Note at which volumes the catalase and salt elute. The catalase is weakly coloured but may also be detected with H₂O₂ like you did in experiment 1.

Results and discussion points that should be included in your lab report
1. In which order are the molecules eluted?
2. When do you think it is useful to make a desalting of the sample during a purification procedure?
3. Suggest an alternative method to remove the salt.

Experiment 3. Separation of two proteins by ion exchange chromatography
In this experiment a small ion exchange column is packed and subsequently used for the separation of catalase and cytochrome c.

• Pasteur pipette and a small piece of glass wool.
• 8-10 small test tubes
• 1-2 ml of DEAE-Sepharose
• Buffer A: 15-20 ml 25 mM Tris HCl pH 8.0
• Buffer B: 5 ml 25 mM Tris HCl pH 8.0 containing 0.2 M NaCl.
• Catalase (1 mg/ml) and cytochrome c (1 mg/ml)

1. Put a small piece of glass wool in the Pasteur pipette and mount it in a stand. Put a beaker under the column outlet. The glass wool will stop the gel from being rinsed out of the column.
   
   
2. Add carefully approximately 1 ml of DEAE-Sepharose to the pipette. You avoid air bubbles if you apply the solution along the wall of the pipette. Equilibrate the column with 3 column volumes of 25 mM Tris HCl pH 8.0.
   
   
3. Place the column over a rack like in exp. 2 and apply 0.5 ml of a solution containing catalase (1 mg/ml) and cytochrome c (1 mg/ml).
   
   
4. Elute stepwise 1.0 ml each time with 25 mM Tris HCl pH 8.0 until the eluate is colourless. Then elute with approx. 3 x 1 ml of 25 mM Tris HCl pH 8.0 containing 0.2 M NaCl until the second protein elutes. Cytochrome c is clearly coloured and catalase may be detected as in earlier experiments.

Results and discussion points that should be included in your lab report
1. Which protein binds stronger?
2. What can be said about the isoelectric point for each protein?
3. Imagine that you use an cation exchanger at the same pH. How would that influence the elution of the proteins?
4. Could these proteins have been separated by some other method?

Experiment 4. Affinity chromatography - Binding of trypsin to the inhibitor benzamidine
Trypsin is an enzyme belonging to the serine proteinase family. Several trypsin inhibitors have been characterised and one of them is benzamidine (Figure 1). In the following experiment benzamidine has been covalently linked to Sepharose beads and this column material has been packed into small pre-packed columns. If you apply a solution containing trypsin to this column material the protein should reversibly bind to the ligand benzamidine and later be recovered by either a pH change or addition of excess free ligand to elute the protein. In our case we will decrease the pH to elute the protein.

Figure 1. Partial structure of Benzamidine Sepharose.


To detect trypsin we will use an artificial substrate p-nitrophenyl-p'-guanidinobenzoate (NPGB). When this substrate is cleaved a strongly yellow coloured compound (p-nitrophenol) is formed (Figure 2).

Figure 2. Cleavage of the substrate p-nitrophenyl-p'-guanidinobenzoate (NPGB) by trypsin.

• Column: HiTrap Benzamidine Sepharose 4 Fast Flow (1 ml) with Luer lock adapter
• 1 ml syringe
• 12 small test tubes
• Buffer A = Binding buffer: 15-20 ml 50 mM Tris-HCl pH 8.0 containing 0.5 M NaCl
• Buffer B = Elution buffer: 10 ml 50 mM Glycine-HCl pH 3.0.
• Sample: 0.5 ml trypsin (10 mg/ml)
• 1 ml NPGB (1.0 mg/ml, freshly made!)
• 1 ml 1 M TrisHCl pH 8.0
• Small beaker with distilled water
• Waste beaker

To avoid contact with buffers and other solutions it is advisable that you wear gloves during this experiment

1. Put 12 test tubes in a rack and number them 1-12.
   Add 100 ml 1 M Tris-HCl pH 8.0 to numbers 7-11 You will later elute the protein by decreasing the pH to 3.0 into these tubes and the extra pH-8-buffer will help keeping the pH at a more physiological level.
   Add 1ml of buffer A to number 12. This will be used as a control tube.
   
   
2. The HiTrap system consists of convenient pre-packed columns that you may run either connected to a pump (e.g. FPLC) or manually with the aid of a syringe. The column is stored in ethanol and you should start by washing it with distilled water. Replace the top lid of the column with a Luer lock connection and remove the bottom nut. Fill a syringe with distilled water, connect it to the top of the column and flush it SLOWLY at a speed of 1 ml/min. A recommended wash volume is 3 ml. All the subsequent solutions are applied like this with the syringe.
   
   
3. Continue to wash the column with 5 ml of buffer A (binding buffer). Now the column is ready to use.
   
   
4. Apply 0.5 ml trypsin to the column (use the syringe speed of ~1 ml/min). Start collecting the eluted fluid in the first tube. Then leave the column with the added protein for 5-10 min to allow the trypsin to bind to its ligand.
   
   
5. Apply 5 x 1 ml of buffer A (binding buffer). Collect in tubes 2-6.
   
   
6. Apply 5 x 1ml of buffer B (elution buffer). Collect in tubes 7-11.
   
   
7. Add 50 ml NPGB to all tubes 1-12. Mix and observe any colour change. Leave the tubes for 5 minutes and observe them again.
   
   
8. Wash the column with 5 ml of water and put back the lids.

1. Does trypsin have an affinity for benzamidine?
2. What happens when you add the low pH buffer?
3. Could you have used the same type of column for any other proteins?

Experiment 5. SDS-polyacrylamide gel electrophoresis
We will separate proteins on a SDS-polyacrylamide gel and use the result to construct a relation between the migration properties on the gel and the molecular weight of the protein subunits. The gels will be precast and polymerized before you start.

• Precast polyacrylamide gel plates, 8-16% in acrylamide
• Electrophoresis running buffer (25 mM Tris HCl, 0.2 M glycine pH 8.8, 0.1% SDS)
• Samples - one per person, micropipettes, tips, gloves
• Sample disruption solution (200 mM Tris HCl pH 6.8, 30% glycerol,
• 0.02% bromphenol blue, 2% SDS, 5 % b-mercapto ethanol)
• Heating block at 90-100° C
• Electrophoresis equipment - buffer chamber and power supply
• Stain solution (5% methanol, 10% acetic acid, 0.1% Comassie Brilliant Blue R250)
• Destain solution (same as the stain solution but without the dye), plastic vessels

1. Mount the precast gel plates in the buffer chamber. You may run 2 gels/chamber which makes it suitable to use one gel per large group of 4-5 persons. Fill the sample wells and buffer chambers with running buffer.
   
   
2. Prepare your samples by adding 25 ml of sample disruption solution to 25 ml of sample. Incubate at 90° C for 5 min. Load the samples on the gel according to instructions. The samples will 'fall' nicely into their wells due to the presence of glycerol which increases the density. Put on the top lid of the chamber.
   
   
3. Connect the chamber to a power supply. The gels should be run at 20 mA/gel. A dye present in the sample solution makes it possible to observe the front moving towards the anode. Normal running time for the gel should be around 60 min.
   
   
4. Switch off the power supply and dismount the gels. WEAR GLOVES AND LAB COAT. The stain solution will stain your clothes a nice shade of deep blue if you spill it. Be careful not to break the gels (or the glass plates). Transfer the gel to a stain solution for 30 min. Then destain in destain solution for at least h. After staining with Coomassie and subsequent destaining, protein bands appear strongly blue.

Data, results and discussion points that should be included in your lab report
1. What conclusions can be drawn about size of the proteins and the way they migrate in the gel?
2. Use the result from a well with a standard mixture of proteins to construct a graph where you plot the migration of the proteins (distance from starting point at the cathode in mm) as a function of the logarithms of their subunit molecular weights. Use the standard curve to determine the denatured molecular weights of your other proteins.
3. Why is SDS present in the gel, samples and buffer?
4. The sample disruption buffer contains b-mercapto ethanol. What is the function of the reductant? Would the pattern of protein bands look different if the reducing agent was omitted from the sample?
5. You have used both gel filtration and SDS-PAGE to determine the molecular weights for some typical proteins. Compare the results and try to deduce whether these proteins are monomers, multimers (e.g. homo or hetero dimers?) in their native states.

Writing lab reports
This wet lab/tutorial consists of five separate experiments, all dealing with the separation (purification) and analysis of proteins. In your lab report, each experiment should be presented separately according to the general guidelines for writing lab reports. Finish the report with a "Conclusions" section where you discuss advantages and disadvantages of the different methods.

Some useful expressions
Here is a short english-swedish dictionary of common protein purification terms

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Lab by Margareta Ingelman
Page updated 2003.02.12 by stefan@xray.bmc.uu.se
Copyright © 1997, . Department of Molecular Biology SLU. All rights reserved.