Protein separation techniques basic electrophoresis Electrophoresis is a

Protein separation techniques – basic electrophoresis Electrophoresis is a powerful technique for protein separation and separated proteins can be visualized after subsequent staining steps. It is based on the principle of migration of charged proteins in an electric field. Harini Chandra Affiliations

1 Master Layout (Part 1) This animation will consist of 2 parts. Protein purification by: Part 1 – Sodium Dodecyl Sulphate (SDS)-PAGE Part 2 – Blue Native (BN)-PAGE SDS-PAGE 2 Buffer Cathode 3 4 Polyacrylamide gel with SDS Anode 5 Buffer reservoir Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook) Stained gel

1 2 3 4 Definitions of the components: Part 1 – SDS-PAGE 1. SDS-PAGE: A protein separation technique based on gel electrophoresis that is carried out under denaturing conditions. Separation in SDS-PAGE occurs almost exclusively on the basis of molecular weight since all proteins have a similar charge-to -mass ratio and shape after they have bound SDS. 2. Sample: In SDS-PAGE, the protein sample to be analyzed is mixed with sodium dodecyl sulphate (SDS), an anionic detergent that causes the protein to denature. Roughly one molecule of SDS binds to every two amino acid residues. Therefore the amount of SDS binding to any protein will be proportional to its molecular weight. This also confers a large negative charge on every protein, thereby causing the original intrinsic charge of the protein to be insignificant. A small amount of reducing agent like dithiothreitol (DTT) or b-mercaptoethanol is also added which reduces the disulphide linkages present in the protein. A tracking dye like bromophenol blue (BPB) which is a small molecule and migrates ahead of other sample proteins is added to know the distance of migration during the course of electrophoresis. 3. Polyacrylamide gel: The gel for SDS-PAGE consists of acrylamide, N, N’methylenebisacrylamide and SDS in a suitable buffer. The molecular dimensions of the pores can be controlled by varying the amount of N, N’-methylenebisacrylamide with free-radical cross-linking being facilitated by APS and TEMED. 4. Well: These are small indentations made in the gel into which the sample is loaded. Usually, several samples can be run in parallel lanes simultaneously. 5

1 2 Definitions of the components: Part 1 – SDS-PAGE 5. Buffer: A solution that can resist any changes in p. H is known as a buffer. For electrophoresis, a buffer solution that can maintain the protein molecules with a net negative charge is placed in both upper and lower reservoirs of the electrophoresis unit. The negative charge ensures that all molecules migrate in one direction towards the anode in the lower reservoir. 6. Cathode and Anode: The oppositely charged electrodes to which current is applied. The negatively charged protein molecules migrate towards the positive anode. 3 4 5 7. Stained gel: After the gel has been run, the separated bands can be viewed by several methods. Staining them with an appropriate dye is the most common technique for visualizing proteins. Coomassie brilliant blue is one of the most widely used dyes. However, another sensitive technique is silver staining which allows proteins to be detected in nanogram quantities.

1 Part 1, Step 1: Gel casting Buffer 2 Gel containing SDS 3 4 5 Polyacrylamide gel cast for SDS-PAGE Electrophoresis unit Action The regions marked as ‘gel’ and ‘buffer’ must be filled with a light blue colour. Description of the action (Please redraw all figures. ) The figure on the left must be shown first. Then, the regions marked as ‘gel’ and ‘buffer’ must be filled with a light blue colour. Audio Narration The polyacrylamide gel containing SDS is cast between glass plates as a vertical slab in the same buffer that is used for electrophoresis. The molecular dimensions of the pores can be controlled by varying the amount of N, N’methylenebisacrylamide with free-radical crosslinking being facilitated by APS and TEMED. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

1 Part 1, Step 2: Well comb Sample well creation Sample wells 2 3 4 5 Action The marked ‘sample wells’ must be made in the gel as shown. Description of the action (Please redraw all figures. ) The comb must be moved downward in the direction shown. It must rest in the blue region for a couple of seconds and then must be moved back upwards as shown in the third figure. Once it is moved upwards, the sample wells must appear. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook) Audio Narration Sample wells of uniform size, shape and separation are made using a comb which is placed in the gel as soon as it has been poured. After the gel has polymerized, the comb is removed, providing a gel ready for the process.

1 Part 1, Step 3: Sample preparation Negatively charged SDS 2 3 SDS, DTT & BPB added Sample heating Protein sample 4 5 Action The green solution must be added to the grey solution in the tube followed by animations as shown Folded proteins Description of the action (Please redraw all figures. ) First the green solution must be added to the grey solution in the tube, which must then turn green. The grey oval below the tube must then turn red to indicate heating. Sample must be zoomed into and figures inside the circle must be shown Denatured proteins bound to SDS Audio Narration SDS is a negatively charged anionic detergent that binds to protein molecules and causes them to denature. The DTT used breaks any disulphide linkages that may be present. The binding of SDS causes the proteins to have a uniform charge-tomass ratio, thereby allowing separation purely on the basis of molecular weight.

Part 1, Step 4: 1 Sample loading Loaded well 2 3 4 5 Protein sample mixed with SDS and DTT Action The hand should move from tube to each of the sample wells next with each one being coloured blue as it moves. Description of the action (Please redraw all figures. ) First show the figure on the left with the hand moving into the tube and coming out. Then show the hand moving into the first well as shown in the middle panel. The well must be filled with a blue colour as in the third panel. The hand must then be moved back up and into the second well. The second well must similarly be filled with the blue colour. This must continue until all wells are filled up. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook) Audio Narration The protein samples are loaded into the wells with the help of a micropipette.

1 Part 1, Step 5: Sample electrophoresis Connected to supply 2 Direction of migration 3 4 5 Buffer in reservoir Action Description of the action The wavy lines must move as shown in the figure. The purple line must move farthest followed by red and then green. (Please redraw all figures. ) First show the figure on the left. The figure must be zoomed into and the first blue square must appear. The lines shown on the top must move slowly in the direction indicated to the positions shown in the second blue square. The lines must then again move further until the third blue square positions are reached. Audio Narration Once the samples have been loaded, a direct current supply between 100 -350 V is passed depending upon the size of the gel for a time sufficient to separate the protein mixture into discrete bands based on their molecular weight. Progress of electrophoresis can be observed with the help of tracking dye. The larger proteins are retarded in the gel and remain close to the point of application while the smaller proteins migrate further along the gel. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

Part 1, Step 6: 1 Gel staining and viewing Silver stained gel 2 High molecular weight proteins 3 4 5 Coomassie stained gel Low molecular weight proteins Action Description of the action This figure should appear (Please redraw all figures. ) The figure with all its labels must appear. Audio Narration The gel is then stained with either Coomassie or silver and viewed to observe the various discrete protein bands.

1 Master Layout (Part 2) This animation will consist of 2 parts. Protein purification by: Part 1 – Sodium Dodecyl Sulphate (SDS)-PAGE Part 2– Blue Native (BN)-PAGE BN-PAGE 2 Buffer 1 Cathode 2 3 4 5 3 4 Polyacrylamide gel ( with TEMED & APS) Anode Buffer reservoir 5 Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook) Stained gel 6 7

1 2 3 4 5 Definitions of the components: Part 2 – BN-PAGE 1. BN-PAGE: Native PAGE is an electrophoretic separation technique that is carried out under non-denaturing conditions and separates proteins on the basis of their net charge and size. Blue native (BN) PAGE is a variation of native PAGE technique where Coomassie blue dye is added to the sample to provide the necessary charge to the protein complexes for migration and separation in the gel. Unlike SDS however, Coomassie does not denature the protein sample. It is often used to determine molecular weight of native proteins and is extremely useful for single step isolation of proteins from cell and tissue homogenates. 2. Sample: This consists of the protein mixture that needs to be separated based on their net charge and size. The sample is mixed with Coomassie which gives the proteins the necessary charge for separation in the gel. In order to prevent the sample from mixing with the buffer solution, it is usually dissolved in a minimal amount of dense material like glycerol or sucrose solution. A tracking dye like bromophenol blue (BPB) which is a small molecule and migrates ahead of other sample proteins is added to know the distance of migration during the course of electrophoresis. 3. Polyacrylamide gel: It is one of the most commonly used gels for electrophoretic separations and is prepared by free radical-induced polymerization of acryl-amide and N, N’-methylenebisacrylamide (cross-linking agent) in a suitable buffer. Ammonium persulfate (APS) and tetramethylenediamine (TEMED) are added to facilitate generation of free radicals and cross-linking. The molecular dimensions of the pores can be controlled by varying the amount of N, N’-methylenebisacrylamide. The separation is therefore, carried out according to both the size and charge of the protein such that large molecules are retained in the gel more than smaller ones.

1 2 3 4 5 Definitions of the components: Part 2 – BN-PAGE 4. Well: These are small indentations made in the gel into which the sample is loaded. Usually, several samples can be run in parallel lanes simultaneously. 5. Buffer: A solution that can resist any changes in p. H is known as a buffer. For electrophoresis, a buffer solution that can maintain the protein molecules with a net negative charge is placed in both upper and lower reservoirs of the electrophoresis unit. The negative charge ensures that all molecules migrate in one direction towards the anode in the lower reservoir. 6. Cathode and Anode: The oppositely charged electrodes to which current is applied. The negatively charged protein molecules migrate towards the positive anode. 7. Stained gel: After the gel has been run, the separated bands can be viewed by staining them with an appropriate dye. For proteins, the most commonly used dye is Coomassie brilliant blue. The gel is soaked in an acidic, alcoholic solution of the dye, which denatures the protein and fixes it in the gel and also complexes the dye to the protein.

1 Part 2, Step 1: Gel casting Buffer 2 Gel 3 4 5 Polyacrylamide gel cast for electrophoresis Electrophoresis unit Action Description of the action The regions marked as ‘gel’ and ‘buffer’ must be filled with a light blue colour. (Please redraw all figures. ) The figure on the left must be shown first. Then, the regions marked as ‘gel’ and ‘buffer’ must be filled with a light blue colour. Audio Narration The polyacrylamide gel is cast between glass plates as a vertical slab in the same buffer that is used for electrophoresis. The gel is prepared by free radical-induced polymerization of acryl-amide and N, N’-methylenebisacrylamide in a suitable buffer. Ammonium persulfate (APS) and tetramethylenediamine (TEMED) are added to facilitate generation of free radicals and cross-linking. The molecular dimensions of the pores can be controlled by varying the amount of N, N’-methylenebisacrylamide. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

1 Part 2, Step 2: Well comb Sample well creation Sample wells 2 3 4 5 Action The marked ‘sample wells’ must be made in the gel as shown. Description of the action (Please redraw all figures. ) The comb must be moved downward in the direction shown. It must rest in the blue region for a couple of seconds and then must be moved back upwards as shown in the third figure. Once it is moved upwards, the sample wells must appear. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook) Audio Narration Sample wells of uniform size, shape and separation are made using a comb which is placed in the gel as soon as it has been poured. After the gel has polymerized, the comb is removed, providing a gel ready for the process.

1 Part 2, Step 3: Sample preparation Negatively charged SDS 2 3 Coomassie blue dye Protein sample 4 5 Action The green solution must be added to the grey solution in the tube followed by animations as shown Folded proteins Description of the action (Please redraw all figures. ) First the blue solution must be added to the grey solution in the tube, which must then turn blue. Sample must be zoomed into and the figure in first circle must be shown. The circles with negative charge must then be shown to bind to the green & purple ‘proteins’ as depicted in second circle. Native proteins bound to Coomassie Audio Narration The protein sample, present in a suitable buffer system, is mixed with the Coomassie blue dye which provides the necessary charge to the protein complexes thereby facilitating their separation in the gel. Unlike SDS, the dye does not denature the proteins but binds to them in their native state.

Part 2, Step 4: 1 Sample loading 2 3 4 5 Protein sample Action The hand should move into the tube and then into each well with each one being coloured blue as it moves. Description of the action (Please redraw all figures. ) First show the figure on the left with the hand moving into the tube and coming out. . Then show the hand moving into the first well as shown in the middle panel. The well must be filled with a blue colour as in the third panel. The hand must then be moved back up and into the second well. The second well must similarly be filled with the blue colour. This must continue until 7 of the 10 wells are filled up. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook) Audio Narration The protein samples are then loaded into the wells with the help of a micropipette.

1 Part 2, Step 5: Sample electrophoresis Connected to supply 2 Direction of migration 3 4 5 Buffer in reservoir Action Description of the action The small lines shown in the blue squares on the right must be shown to move in the direction indicated. (Please redraw all figures. ) First show the figure on the left. The figure must be zoomed into and the first blue square must appear. The lines shown on the top must move slowly in the direction indicated to the positions shown in the second blue square. The lines must then again move further until the third blue square positions are reached. Audio Narration Once the samples have been loaded, a direct current supply of around 100 -350 V is passed depending upon the size of the gel for a time sufficient to separate the protein mixture into discrete bands based on their charge-to-mass ratio. Progress of electrophoresis can be observed with the help of tracking dye. The larger proteins are retarded in the gel and remain close to the point of application while the smaller proteins migrate further along the gel. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

Part 2, Step 6: 1 Gel staining and viewing 1 2 2 3 4 Gel stained with Coomassie brilliant blue. 3 4 5 5 6 7 High molecular weight proteins Low molecular weight proteins Action Description of the action This figure should appear (Please redraw all figures. ) The figure with all its labels must appear. Audio Narration The gel is then stained with Coomassie brilliant blue and viewed to observe the various discrete protein bands.

1 2 3 Interactivity option 1: Step No: 1 Three tubes having protein samples containing insulin, myglobin and hemoglobin are present. Insulin is a 51 amino acid peptide hormone having a molecular weight of 5. 8 k. D. It is composed of two chains linked by disulphide bonds and plays a very important role in maintenance of blood glucose levels. Myoglobin is an oxygenbinding, globular protein composed of a single polypeptide chain having 154 amino acid residues with a molecular weight of 16. 7 k. D. It is made up of 8 alpha helices and is structurally related to hemoglobin. Hemoglobin is an iron containing, oxygentransport protein. It is composed of four subunits (2 a and 2 b) linked together by salt bridges and other hydrophobic interactions with a mass of 68 k. D. How can one determine which tube contains which protein and then estimate the number of subunits present in each protein? 1. Run all three samples by BN-PAGE. 4 3. Run all three samples by both BN-PAGE and SDSPAGE. Interactivity Type 5 2. Run all three samples by SDS-PAGE. Click on any of the three option tabs. Options User should be allowed to choose any one of the options. If he chooses 1 or 2, they should turn red but they can try again. If user chooses the third option, it should turn green. Boundary/limits Results If user chooses option 1 or 2, it should turn red with a ‘incorrect method’ error message. The user can try until he clicks the correct answer. If user chooses option 3, it should turn green and user should be redirected to sample animation slides (Steps 2 a-d)

1 2 Interactivity option 1: Step No: 2 (a) A comparison of the electrophoretic separation profiles of hemoglobin, insulin and myoglobin using both BN-PAGE and SDS-PAGE will allow one to determine which tube contains which protein sample and how many subunits may be present in each protein. Insulin: Has two polypeptide chains of different mass held together by disulphide linkages. 3 4 5 Hemoglobin (Hb): Tetramer consisting of 2 a and 2 b subunits, held together by salt bridges, hydrophobic interactions and hydrogen bonds. Myoglobin (Mb): It is a single chain globular protein having eight alpha helices.

1 2 Interactivity option 1: Step No: 2 (b) A comparison of the electrophoretic separation profiles of hemoglobin, insulin and myoglobin using both BN-PAGE and SDS-PAGE will allow one to determine which tube contains which protein sample and how many subunits may be present in each protein. BN-PAGE SDS-PAGE Samples loaded as such Samples mixed with SDS and DTT 3 4 5 Native polyacrylamide gel Polyacrylamide gel with SDS Buffer in reservoir

1 2 Interactivity option 1: Step No: 2 (c) A comparison of the electrophoretic separation profiles of hemoglobin, insulin and myoglobin using both BN-PAGE and SDS-PAGE will allow one to determine which tube contains which protein sample and how many subunits may be present in each protein. SDS-PAGE BN-PAGE 1 3 4 5 2 3 Direction of migration 1 2 3

1 2 Interactivity option 1: Step No: 2 (d) A comparison of the electrophoretic separation profiles of hemoglobin, insulin and myoglobin using both BN-PAGE and SDS-PAGE will allow one to determine which tube contains which protein sample and how many subunits may be present in each protein. BN-PAGE 1 2 3 4 Completed gels stained with Coomassie blue. SDS-PAGE 1 2 3 4 Hb (68 k. D) 3 MW marker, Pepsin, (35 k. D) Hb (a-subunit) MW marker, Pepsin, (35 k. D) Mb (16. 7 k. D) Hb (bsubunit) Mb Insulin (5. 8 k. D) Insulin A chain 4 5 Since the molecular weight of each protein is known, the migration distance will indicate which band corresponds to which protein. More than 1 band will appear for Hb and insulin indicating that they have multiple subunits. The thicker bands are indicative of more than one subunit of nearly identical molecular mass. Insulin B chain

1 2 Interactivity option 2: Step No: 1 Which technique would be suitable for determination of molecular weight of the enzyme a-amylase? Although this technique may be used, it may not always be very accurate since it also depends on net charge of the protein. 1. BN-PAGE 3 4 5 2. SDS-PAGE Interacativity Type Click on any of the two option tabs. Options User should be allowed to choose any one of the options. If he chooses 1, it should turn red and the message displayed must be shown. If user chooses 2, it should turn green and user must be redirected to steps 2(a)(c) Boundary/limits Results If user chooses option 1, it should turn red and the message shown must be displayed. The user can then choose the other option and be redirected to steps 2(a)-(d). If user chooses option 2, it must turn green and he is redirected to steps 2(a)-(d).

1 2 3 4 5 Interactivity option 2: Step No: 2 (a) Determination of molecular weight of a-amylase: SDS-PAGE is commonly used for determination of molecular weight of an unknown protein by running it along with other protein markers of known molecular weight. Protein Molecular weight, M (k. D) Bovine serum albumin (BSA) 66. 4 Lysozyme 14. 7 Pepsin 35 Papain 38. 9 a-amylase ? Samples mixed with SDS and DTT Polyacrylamide gel with SDS Sample loading Buffer in reservoir

1 2 Interactivity option 2: Step No: 2 (b) Determination of molecular weight of a-amylase: SDS-PAGE is commonly used for determination of molecular weight of an unknown protein by running it along with other protein markers of known molecular weight. Connected to supply 1 Protein markers 2 a-amylase 3 Direction of migration 4 5 Sample electrophoresis

1 2 Interactivity option 2: Step No: 2 (c) Determination of molecular weight of a-amylase: SDS-PAGE is commonly used for determination of molecular weight of an unknown protein by running it along with other protein markers of known molecular weight. 1 2 BSA a-amylase 3 a-amylase Papain Pepsin Lysozyme 4 5 Lysozyme Completed gel stained with Coomassie blue. Mobility of the unknown protein can therefore be used to determine its molecular weight from a graph as shown above. Based on this, molecular weight of a-amylase is 53 k. D.

1 Questionnaire 1. Four proteins W, X, Y and Z have molecular weights of 25. 4 k. D, 80. 2 k. D, 30. 5 k. D and 44. 3 k. D respectively. Which of them will migrate the farthest when run by SDS-PAGE? 2 Answers: a) X b) Y c) W d) Z 2. If the p. H of the electrophoresis buffers are modified to have a p. H of 2. 3, then what would happen to the proteins? They would 3 Answers: a) Migrate towards anode b) Migrate towards cathode d) Get denatured c) Remain stationary 3. How many bands would appear for a protein having 3 subunits linked together by disulphide bonds in native and SDS-PAGE? 4 5 Answers: a) 1 and 3 b) 3 and 1 c) 3 and 3 d) 1 and 1 4. The relative electrophoretic mobilities of a 45 k. D and a 70 k. D protein on SDS-PAGE are respectively 0. 7 and 0. 3. What would be the molecular mass of a protein with relative mobility of 0. 44? Answers: a) 68 k. D b) 54 k. D c) 80 k. D d) 60 k. D

1 Questionnaire 5. Match the protein with the number of subunits they contain. 2 1. Myglobin i) 2 2. Hemoglobin ii) 1 3. Insulin iii) 4 Answers: a) 1 -ii, 2 - iii, 3 -i b) 1 -i, 2 -ii, 3 -iii c) 1 -iii, 2 -ii, 3 -i d) 1 -i, 2 -iii, 3 -ii 3 4 5

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: Pyndiah, S. et al. , Two-dimensional Blue Native/SDS gel electrophoresis of multiprotein complexes from Helicobacter pylori. Mol. Cell. Proteomics 2007, 6, 193 -206. Camacho-Carvajal, M. M. et al. , Two-dimensional Blue Native/SDS Gel Electrophoresis of Multi-Protein Complexes from Whole Cellular Lysates. Mol. Cell. Proteomics 2004, 3, 176 -182. Wittig, I. , Braun, H. P. , Schägger, H. Blue native PAGE. Nat. Protoc. 2006, 1(1), 418 -428.