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Protein separation techniques: two dimensional gel electrophoresis Harini Chandra Affiliations Electrophoresis is a powerful technique for finer protein.

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Presentation on theme: "Protein separation techniques: two dimensional gel electrophoresis Harini Chandra Affiliations Electrophoresis is a powerful technique for finer protein."— Presentation transcript:

1 Protein separation techniques: two dimensional gel electrophoresis Harini Chandra Affiliations Electrophoresis is a powerful technique for finer protein separation and visualization of separated proteins. It is based on the principle of migration of charged proteins in an electric field. Electrophoretic techniques have developed significantly for improved protein separation and detection.

2 Master Layout (Part 1) This animation will consist of 2 parts. Protein purification by: Part 1 – Two dimensional electrophoresis (2-DE) Part 2 – Difference Gel Electrophoresis (DIGE) 2-D Electrophoresis First dimension : Isoelectric focusing Increasing pI IPG strip SDS- polyacrylamide gel Second dimension: SDS-PAGE Decreasing molecular weight Increasing pI Source: Biochemistry by A.L.Lehninger, 4 th edition (ebook)

3 Definitions of the components: Part 1 – 2-D electrophoresis Two dimensional electrophoresis (2-DE): 2-DE is a powerful electrophoresis separation technique that separates proteins in two directions. Isoelectric focusing is carried out in the first dimension which separates proteins on the basis of their unique isoelectric points. This is followed by SDS-PAGE which separates proteins on the basis of their molecular weights. 2. Isoelectric focusing (IEF): This is a technique for separation of proteins based on their isoelectric points and constitutes the first dimension of separation in 2-DE. The pH at which a protein has no net charge and therefore does not migrate in an electric field is called its isoelectric point (pI). Every protein has a unique pI due to their different amino acid compositions. When a protein mixture is loaded on to a gel strip having a pH gradient and an electric field is applied, every protein migrates until it reaches the pH corresponding to its pI. 3. IPG strip: Commercially available immobilized pH gradient (IPG) gel strips have replaced tube gels and have considerably facilitated the process of isoelectric focusing by eliminating the tedious steps of gel preparation and pH gradient establishment using ampholyte solutions. These strips, available across the pH range, contain a preformed pH gradient immobilized on a precast polyacrylamide gel placed on a plastic support. Narrow pH ranges can be selected for fine separations while broader pH ranges are also available for crude separations. These strips only need to be rehydrated with a suitable buffer before use.

4 Definitions of the components: Part 1 – 2-D electrophoresis SDS-polyacrylamide gel: It is one of the most commonly used gels for separation of proteins based on their molecular weights. It is prepared by free radical induced polymerization of acryl-amide and N, N’-methylenebisacrylamide, facilitated by APS and TEMED. The gel also contains the anionic detergent, sodium dodecyl sulhpate (SDS), and a reducing agent like dithiothreitol (DTT) or  -mercaptoethanol. These are responsible for denaturing the protein and facilitate their separation solely on the basis of size. 5. SDS-PAGE: The second dimension of separation in 2-DE is SDS-PAGE, which separates proteins based on their mass. Denatured proteins of high molecular weight do not migrate far in the gel and remain close to the point of application. However, the low molecular weight proteins can easily make their way through the gel pores and therefore move a greater distance in the gel.

5 Part 1, Step 1: ActionAudio Narration Description of the action As shown in animatio n. First show the blue solution in the tray followed by the strip being placed in it. Next show the strip being removed and placed in the gray lane of the indented rectangle. Next the tube must appear and the orange liquid must be added across the strip as shown. Prior to isoelectric focusing in 2-DE, the commercially available IPG strips must be rehydrated. This is done by soaking them for hours in the protein sample which is contained in a suitable buffer solution. Once the strips are rehydrated, they are covered with mineral oil to prevent evaporation of solution and drying of the gel. 2-DE, Isoelectric focusing Buffer solution with protein sample IPG strip Mineral oil Addition of mineral oil to avoid evaporation Strip rehydration, h

6 Part 1, Step 2: ActionAudio Narration Description of the action As shown in animatio n. First show the grey indented rectangle. Next show the blue strip being place in one lane with the red arrow and labels. Next, the circles must appear with the labels. Next show the ‘sample’ tube on the right and the hand moving into the tube and then into the circle in the second lane as depicted. Next, the hand must move into the orange tube and add solution into the lane in the second circle. Once the IPG strips have been rehydrated, the strip is placed in the tray and the sample is added through the sample cup followed by the cover fluid to prevent drying of gel. In passive loading, the gel strip is placed face down in the cover fluid containing the sample for hours after which it is run in an electric field. 2-DE, Isoelectric focusing pH 3 pH 9 IPG strip Active sample loading Protein sample Sample cups IPG strip tray Cover fluid (to prevent drying of gel)

7 Part 1, Step 3: ActionAudio Narration Description of the action The grey bands must be shown to move in the direction indicated. (Please redraw instrument) The instrument on top must appear followed by the dotted lines. The strip on the left must appear and the grey bands must be shown to move in the direction indicated until they come to rest at the positions shown in the right-most figure. These bands must then change colour to blue. These loaded strips are then focused on an isoelectric focusing unit by passing current. The various proteins of the sample mixture migrate in the electric field and come to rest when the pH is equal to their pI i.e. they become neutral and are no longer affected by the electric field. Progress of electrophoresis is monitored by means of a tracking dye like bromophenol blue (BPB) which is a small molecule and therefore migrates ahead of all other proteins. Direction of migration Progress of electrophoresis is followed by means of tracking dye. Proteins are separated along the pH gradient according to their respective pI. 2-DE, Isoelectric focusing Isoelectric focusing unit Tracking dye

8 Part 1, Step 4: ActionAudio Narration Description of the action As shown in animation. First show the strip in the light blue solution in the first vessel. Next this strip must be transferred to the dark blue solution in the next vessel. The strip must then be transferred into the slot in the slab on the right as depicted in the animation. The IPG strip is equilibrated in a reducing agent like DTT followed by an alkylating agent, iodoacetamide which prevents reformation of the reduced bonds. This strip containing the separated proteins is then placed on the SDS-polyacrylamide gel slab for further protein separation in the second dimension based on their molecular weight. 2-DE, SDS-PAGE The IPG strip is then placed on the SDS-polyacrylamide gel for separation in the second dimension. SDS-polyacrylamide gel slab Source: Biochemistry by A.L.Lehninger, 4 th edition (ebook) IPG strip 1 st equilibration – gel placed in DTT 2 nd equilibration - gel placed in iodoacetamide

9 Part 1, Step 5: ActionAudio Narration Description of the action The small blue dashes must move in the direction indicated. (Please redraw instrument) First show the electrophoresis unit picture at the bottom followed by dotted lines. Then show the figure on left and the direction of migration. Then show the small blue dashes moving in that direction until they come to rest at the positions indicated in the figure on the right. The proteins on the IPG strip are then subjected to SDS-PAGE by applying a direct current between V depending upon the size of the gel. Any proteins that may have been present as a single band on the IPG strip due to similar isoelectric points can now be separated on the basis of their molecular weight with smaller proteins migrating farthest. 2-DE, SDS-PAGE Final 2-D gel pattern after SDS-PAGE. Decreasing molecular weight Increasing pI Source: Biochemistry by A.L.Lehninger, 4 th edition (ebook) Gel electrophoresis unit

10 Part 1, Step 6: ActionAudio Narration Description of the action View of a sample gel which has been run by 2- DE and stained with Coomassie blue. Each spot provides information about the MW and pI of the proteins. Show image. Spot analysis: MW and pI of protein pH 4pH 7 Increasing pI Decreasing molecular weight Molecular weight markers

11 Master Layout (Part 2) This animation will consist of 2 parts. Protein purification by: Part 1 – Two dimensional electrophoresis (2-DE) Part 2 – Difference Gel Electrophoresis (DIGE) Cy3 Cy2 Cy5 Sample 1 Internal pooled standard 1+2 Sample 2 Mix 2-D electrophoresis Cy3 excitation Cy5 excitation Cy2 excitation Protein samples Cyanine dyes Gel viewing

12 Definitions of the components: Part 2 – Difference gel electrophoresis Sample 1 & sample 2: The protein samples that need to be run and separated on a single gel by labelling them with differently fluorescing dyes. 2. Cyanine dyes (Cy3, Cy5 & Cy2): They are derivatives of N-hydroxy succinimide that covalently bind the  -amino groups of a protein’s lysine residues and are spectrally resolvable as they fluoresce at distinct wavelengths. The labelled samples can therefore be mixed and run on a single gel, thereby eliminating the problem of gel-to-gel variations. 3. Internal pooled standard (1+2): This consists of equal amounts of all samples being run in the experiment, labelled with a Cy2 dye. This pooled sample serves as a useful internal standard for all protein spots on the gel and can be used for normalization of the spots, thereby further reducing any variations that may arise D electrophoresis: The different protein samples are mixed together and run by 2-DE on a single gel, which consists of isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. The proteins are separated according to their isoelectric points and then their molecular weights.

13 Definitions of the components: Part 2 – Difference gel electrophoresis Gel viewing: The gel can be viewed by illuminating it alternately at the excitation wavelengths of each of the cyanine dyes. The Cy3 wavelength illumination will only show those proteins containing the Cy3 dyes, the Cy5 wavelength will only highlight the proteins bonded to the Cy5 dyes while the Cy2 illumination will show all the protein samples of the internal standard. The excitation and emission wavelengths of Cy3 dye are 550 nm and 570 nm respectively while the Cy5 dye has excitation and emission wavelengths of 650 nm and 670 nm.

14 Part 2, Step 1: ActionAudio Narration Description of the action The hand must take solution out of the first and second tubes and put it in the third tube. The hand must be made to move into the tube labled as ‘sample1’ and when it comes out, the level of liquid should decrease slightly. The hand must then move to the ‘internal standard’ tube and the level in that tube must rise slightly. The same protocol must be repeated for tube labeled ‘sample 2’. The pooled internal standard for DIGE is prepared by mixing equal amounts of all samples that are being run in the experiment. This prevents problems of gel-to-gel variations. Internal standard preparation Sample 1 Sample 2 Internal standard

15 Part 2, Step 2: ActionAudio Narration Description of the action The red, blue and green stars must be shown to mix with their respective tubes to give the coloured tubes. The tubes in the first row must appear with their labels. The red, blue and green coloured stars must then be shown to enter one tube each and mix with the grey portion at the bottom. The coloured solution at the bottom must then appear. Each protein sample as well as the internal standard is labeled with a differently fluorescing cyanine dye which allows all protein samples to be simultaneously run on a single gel. The dye binds covalently to the  -amino group of lysine residues in proteins. Dye labeling Sample 1 Sample 2 Internal standard Cy3 Cy5Cy2

16 Part 2, Step 3: ActionAudio Narration Description of the action Show the three coloured tubes mixing together into one tube shown below followed by the micropipette being dipped inside and the yellow tip turning purple. The coloured tubes must appear and then merge to give the coloured tube at the bottom. The micropipette must be dipped in the purple solution and the yellow tip must become purple. It must then move to the blue strip and a small purple layer must appear on top of the circle indicated. There must be formation of bands in the strip after which it must be placed in the groove of the blue slab on the right. The small lines must then migrate from the top in the direction indicated. The labelled protein samples are mixed and run on a single 2-DE gel. Separation takes place on the basis of isoelectric points of the proteins in one dimension and based on molecular weight of the proteins in the second dimension with the smaller proteins migrating further along the gel. Mixing and 2-DE Sample 1 Sample 2 Internal standard Micropipette Mixed IPG strip

17 Part 2, Step 4: ActionAudio Narration Description of the action The red triangle must be shown followed by the blue square with red dots. Then the blue triangle must be shown with the blue square and blue dots. Finally the green must be shown. The red triangle followed by the blue square with red dots must be shown. Next the blue triangle with followed by the blue square with blue dots must be shown. Finally, the green triangle with the blue square and green dots must be shown. The gel containing all the protein samples can be viewed by illuminating it alternately with excitation wavelengths corresponding to the various Cyanine dyes. Gel viewing Cy5 excitation wavelength Only Cy3 labeled proteins Only Cy5 labeled proteins All proteins with Cy2 label Cy2 excitation wavelength Cy3 excitation wavelength DIGE Imager

18 Step 5: ActionAudio Narration Description of the action View of a superimposed DIGE gel depicting all protein spots of multiple samples. Information on molecular weight and pI of proteins can be obtained from these spots. Superimposed DIGE image pH 4pH 7 Increasing pI Decreasing molecular weight

19 Interactivity option 1:Step No: 1 Boundary/limitsInteracativity Type Options Results Which of these two would be the better technique to separate serum protein samples obtained from 250 patients in a clinical trial? 2-D gel electrophoresis (2-DE) Difference gel electrophoresis (DIGE) Click on any of the two option tabs. User should be allowed to choose any one of the options and should be redirected to step 2 if they choose 2- DE or else to step 3 if they choose DIGE. For each option, there is an explanation on whether it is the correct choice or not.

20 Interactivity option 1:Step No: DE, although a very useful technique, may not be the best option in this case for analyzing serum proteins from a large number of patients as it would involve running several individual gels which would be a time consuming process. Also variations across the gels would make comparison of results a problem. Multiple serum samples from patients Blood sample collection Several 2-D gels run in order to analyze all patient samples

21 Interactivity option 1:Step No: 3 (a) DIGE is an extremely valuable tool for analysis of a large number of samples simultaneously without having to overcome the problem of gel-to-gel variation. The control and test samples can be differentially labelled using the cyanine dyes and run on a single gel. Cy3 Mix Multiple serum samples from patients Single 2-D gel analysis Control Control samples Test samples Internal standard Cy5 Test Internal standard Cy2

22 Interactivity option 1:Step No: 3 (b) Serum control sample Serum test sample All serum proteins Illumination of gel under suitable wavelength DIGE is an extremely valuable tool for analysis of a large number of samples simultaneously without having to overcome the problem of gel-to-gel variation. The control and test samples can be differentially labelled using the cyanine dyes. All samples can be completed with half the number of gels compared to 2-DE! DIGE Imager

23 Questionnaire 1. The number of spots shown in a Cy2 labelled internal standard gel is 100 while that of Cy3 labelled protein sample A is 47 in a DIGE experiment. How many spots should be present in the gel having Cy5 labelled protein sample B? Answers: a) 51 b) 55 c) 53 d)‏ The net charge of a protein when it comes to rest on an IEF gel is Answers: a) Positive b) Neutral c) Negative d) Doubly positive 3. Purified monoclonal IgG was subjected to electrophoresis. The number of bands visible by SDS-PAGE, isoelectric focusing and native PAGE are respectively: Answers: a) 2, 1, 1 b) 1, 1, 1 c) 4, 2, 2 d)‏ 4, 1, 1 4. The maximum emission wavelength of Cy3 is Answers: a) 550 nm b) 670 nm c) 650 nm d) 570 nm

24 Links for further reading Books: Biochemistry by Stryer et al., 5 th edition Biochemistry by A.L.Lehninger et al., 3 rd edition Biochemistry by Voet & Voet, 3 rd edition Research papers: Unlu, M., Morgan, M. E., Minden, J. S. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 1997, 18, Van den Bergh, G. And Arckens, L. Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics. Curr. Opin. Chem. Biotechnol. 2004, 15, Alban, A. et al. A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 2003, 3,


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