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Protein separation techniques – Chromatography

Harini Chandra Affiliations. Protein separation techniques – Chromatography.

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Protein separation techniques – Chromatography

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  1. Harini Chandra Affiliations Protein separation techniques – Chromatography Purified proteins are essential for structural and functional studies. Several chromatographic purification techniques have been developed which separate proteins based on parameters such as size, shape, charge or chemical specificity.

  2. Master Layout Affinity chromatography 1 Chromatography Ion exchange chromatography Gel filtration 2 3 Action Description of the action Audio Narration 4 The three images must appear with each of them being a separate tab. Chromatography refers to a collection of lab techniques that carry out separation of complex mixtures by making use of inherent properties of the components of the mixture. It involves the differential partitioning of molecules between a suitable stationary phase and mobile phase. The most commonly used chromatographic techniques are gel filtration, ion exchange chromatography and affinity chromatography. The main heading of chromatography must appear followed by the 3 images and the remaining headings & figures below. User must be allowed to click on any of them so that he is redirected to the appropriate section. When the user moves the cursor over the sub-headings, the definitions given in the next slide must appear. 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook); Biochemistry by Voet & Voet (3rd edition)

  3. Definitions of the components: (Master layout) 1 1. Chromatography:Chromatography refers to a collection of lab techniques that carry out separation of complex mixtures by making use of inherent properties of the components of the mixture. It involves the differential partitioning of molecules between a suitable stationary phase and mobile phase. The most commonly used chromatographic techniques are gel filtration, ion exchange chromatography and affinity chromatography. 2. Gel filtration chromatography: Gel filtration or size exclusion chromatography separates molecules according to their sizes. Small molecules enter the pores of the stationary phase matrix and move slowly through the column while larger molecules pass in between the pores and are removed quickly from the column. 3. Ion exchange chromatography: This technique makes use of a charged stationary phase that binds and retains molecules from a mixture bearing the opposite charge. Components having the same or neutral charge flow through the column without binding. 4. Affinity chromatography: This separation procedure makes use of specific interactions to purify the molecule of interest. The stationary phase matrix is functionalized with a suitable ligand that will selectively bind to the molecule of interest thereby purifying it. 2 3 4 5

  4. Master Layout (Part 1) 1 This animation will consist of 3 sections. Protein purification by: Part 1 – Gel filtration Part 2 – Ion exchange chromatography Part 3 – Affinity chromatography Gel filtration 2 Mobile phase 3 Protein mixture Size exclusion gel matrix 4 Effluent sample fractions 5 Source: Biochemistry by Voet & Voet (3rd edition)

  5. Definitions of the components (Part 1 – Gel filtration) 1 1. Size exclusion gel matrix: The matrix filling the gel filtration column consists of a highly hydrated polymeric material, commonly dextran, agarose or polyacrylamide, which is porous in nature thereby allowing molecules below a particular specified size to pass through them. 2. Protein mixture: The mixture of unpurified proteins of different sizes which is applied to the top of the column for separation. 3. Mobile phase: Following sample application, the proteins are eluted out of the column by means of a suitable mobile phase that carries the protein out with it. For gel filtration, a salt solution of appropriate strength is commonly used which will not have any effect on properties of the proteins being purified. Solvents or buffer systems are often used in other types of chromatography. 4. Effluentsample fractions: The solution leaving the column is collected in suitably sized fractions. Initial fractions will contain only the mobile phase while latter fractions will have purified proteins. 2 3 4 5

  6. Analogy 1 2 Part 1 (Gel filtration): This process is very similar to separation of small particulate matter from food grains using a sieve. The larger grains remain behind on the sieve while the smaller sand/stone particles pass through the sieve and are removed. In keeping with this, gel filtration technique is also commonly referred to as the “molecular sieve”. 3 4 5

  7. Part 1, Step 1: 1 2 Size exclusion gel matrix 3 Chromatography column Packed gel column Action Description of the action Audio Narration 4 Showing the column being filled with a material. (Please redraw all figures.) First show only empty column on the left. Then show the column being filled with the brown coloured beads as shown on the right. • The column is packed with a hydrated porous gel matrix suitable for the required protein separation. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  8. Part 1, Step 2: 1 Sample loading 2 Packed gel column 3 Porous gel matrix Unpurified protein mixture Action Description of the action Audio Narration 4 The packed column is then loaded with the protein sample containing a mixture of unpurified proteins of different sizes. The porous beads of the gel matrix only allow small molecules inside its pores while larger ones remain outside and travel in between the beads. Pouring the solution from the container on the left into the column on the right. (Please redraw all figures.) First show the ‘unpurified protein mixture’ beaker on the right along with the ‘packed gel column’. Next show this beaker being lifted and its contents being poured into the column on the right. The blue & pink particles be layered above the brown beads as shown. This region must then be zoomed into and the inset on the right must be shown with pink circles present in the beads but blue circles present only outside. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  9. Part 1, Step 3: 1 Mobile phase reservoir Mobile phase (salt solution) 2 Direction of flow 3 Action Description of the action Audio Narration 4 The column is eluted with a salt solution of appropriate concentration. Large proteins that cannot enter the pores of the gel move down through the interstitial spaces at a faster rate and are eluted first. The smaller proteins move in and out of the pores thereby taking longer to be removed from the column. The coloured dots must be separated from each other by moving them at different rates. (Please redraw all figures.) Show the column on the left. The column must be zoomed into and the first figure on the right must then be shown in which all the circles are present at the top. Next the blue circles must move down at a quicker rate than the pink circles. Blue circles must not be allowed to enter the big brown circles while the pink circles can travel inside the big brown circles as well. The circles must move down as shown in the subsequent two images on the right. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  10. Part 1, Step 4: 1 2 Sample collection 3 2 1 1 3 1 1 2 4 2 3 5 4 Action Description of the action Audio Narration Liquid coming out of the column must be collected in the tubes. Fractions of appropriate size are collected and analyzed for their protein content. The largest proteins eluting out first will be present in the earlier fractions while the smaller proteins which elute out later will be present in the later fractions. (Please redraw all figures.) Show an empty tube and the column followed by the tube being filled by a light grey colour liquid. Then the first tube must be moved to the left and a second tube must be filled in a similar way. The third tube must be filled with the grey colour & blue dots as shown. The fourth must be filled with grey solution, few blue dots & some pink dots. Then the last tube must be filled only with pink dots as shown in animation. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  11. Part 1, Step 5: 1 Lowest molecular weight 2 Highest molecular weight Protein concentration UV-Vis spectrophotometer 3 1 2 4 3 5 Collected fractions Volume of eluant 4 Action Description of the action Audio Narration The tubes shown must be moved towards the instrument above one at a time with appearance of the graph. (Please redraw all figures.) The tubes must be moved one at a time towards the instrument which should have a hole in which the tube fits. Every time a tube enters and leaves the hole in the instrument, a part of the graph must simultaneously appear. When tubes 1&2 are analyzed, only a straight line parallel to the x-axis of graph must appear. When the tubes with blue dots are analyzed (tube 3 & 4) the blue hump must appear followed by the pink hump when the tube with pink circles is analyzed. Each fraction is analyzed for its protein content using a UV-Visible Spectrophotometer at 280 nm. A graph of eluant volume versus protein concentration is then plotted. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  12. Master Layout (Part 2) 1 This animation will consist of 3 sections. Protein purification by: Part 1 – Gel filtration Part 2 – Ion exchange chromatography Part 3 – Affinity chromatography Ion exchange chromatography 2 Low salt elution buffer High salt elution buffer 3 Proteinmixture Charged stationary phase 4 Effluent sample fractions 5 Source: Biochemistry by Voet & Voet (3rd edition)

  13. Definitions of the components (Part 2 – Ion exchange chromatography) 1 1. Charged stationary phase: The column stationary phase consists of a positively or negatively charged polymeric matrix which will bind molecules of the opposite charge. Commonly used ion exchangers are negatively charged caboxymethyl-cellulose (CM-cellulose), which is a cation exchanger and positively charged diethylaminoethyl cellulose (DEAE-cellulose), which is an anion exchanger. 2. Protein mixture: The unpurified protein mixture consists of proteins of different net charges that are loaded onto the column. The proteins having charges opposite to that of the stationary matrix will bind to it while the remaining proteins will be eluted from the column. 3. Mobile phase/ Elution buffer: Following sample application, the proteins are eluted out of the column by means of a suitable mobile phase that carries the protein out with it. For ion exchange chromatography, buffer systems of suitable pH are used which will first remove the unbound proteins. The buffer is then changed such that the charge of bound proteins is modified and they are also eluted out of the column. 4. Effluentsample fractions: The solution leaving the column is collected in suitably sized fractions for further analysis. The unbound proteins having same charge as the column matrix will be eluted out in the initial fractions while the bound proteins will be eluted later upon changing the buffer system. 2 3 4 5

  14. Part 2, Step 1: 1 2 3 Packed ion exchange column Chromatography column Action Description of the action Audio Narration 4 Showing the column being filled with a material. (Please redraw all figures.) First show only empty column on the left. The column must then be filled with the coarse brown color. This must be zoomed into and the inset must be shown. The column is packed with a suitable cation or anion exchange resin depending upon the charge of the protein that needs to be bound to the column and purified. Cation exchangers bind to and exchange positively charged ions while anion exchangers bind to negatively charged ions. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  15. Part 2, Step 2: 1 Sample loading Net negative charge - Net positive charge + Large net positive charge + 2 Protein sample loaded Packed ion exchange column 3 + + - - + + + + + + + + - - - - + + - - + + Unpurified protein mixture Action Description of the action Audio Narration 4 (Please redraw all figures.) First show the beaker with colored circles having +ve & -ve charges along with the column on the right. Next, show the contents of this beaker being poured into the column. Once it is poured, a pink layer must appear on top. Pouring the solution from the container on the left into the column on the right. The anion exchange column is loaded with the impure protein sample mixture consisting of positively and negatively charged proteins. The positively charged ions bind to the matrix while the negatively charged ions do not and flow through the column . 5 Source: Biochemistry by Voet & Voet (3rd edition)

  16. Part 2, Step 3: 1 + + + + + + + + + + + + + + + + + + + Mobile phase reservoir + + - - - - - - Low salt elution buffer - - - - - - 2 Direction of flow 3 Action Description of the action Audio Narration 4 The column is first eluted with a buffer solution having low salt concentration or a suitable pH such that the negatively charged molecules are removed from the column while the positively charged molecules remain bound to the anion exchange resin. The blue & violet circles with +ve charge must bind to the grey circles while red circles must not. (Please redraw all figures.) First show only the column on the left with the red, violet & blue ovals. Next, the red box must appear which must be zoomed into and the first figure on the right must be shown. Here all the circles must be towards the top with the red ones at the lowest position. Next, the red circles with negative charge must be shown to move down rapidly while the blue & violet circles must bind to the grey circles. The violet circles must move down slightly faster than the blue as shown in the last figure. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  17. Part 2, Step 4: 1 + + + Mobile phase reservoir High salt elution buffer 2 3 Direction of flow Action Description of the action Audio Narration 4 The blue coloured circles must now be moved downward. • (Please redraw all figures.) • First show the column on the left. • The column must be zoomed into and the figure on the right must then be shown. • The blue dots must be shown to move downward towards the bottom of the column. The buffer solution is then changed to one having a high salt concentration or such that the net pH of the protein is modified and it no longer binds the ion exchange resin. Therefore the bound protein is also eluted out in this way. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  18. Part 2, Step 5: 1 2 3 Sample collection + + - - - + + + + - - - + + + - + - - + 2 4 1 5 1 4 3 2 3 2 1 2 1 1 3 4 Action Description of the action Audio Narration Fractions of appropriate size are collected and analyzed for their protein content. The negatively charged proteins which get eluted first will be present in the initial fractions while the positively charged proteins that bind to the column are eluted in the later fractions. Liquid coming out of the column must be collected in the tubes. (Please redraw all figures.) First show the column on the left with the empty tube below. This must be filled with the grey solution. Next show this tube moving to the left and tube 2 being filled. Next tube 3 must be filled with grey solution & red negative circles. Then tube 4 must be filled with violet +ve circles & finally tube 5 with +ve blue circles. The colored bands in the column must be shown to move down progressively. 5 Source: Biochemistry by Voet & Voet (3rd edition)

  19. Part 2, Step 6: 1 Large negative charge Large positive charge Protein concentration 2 UV-Vis spectrophotometer Volume of eluant 3 + + 2 4 1 5 3 - + + - + + + Collected fractions - 4 Action Description of the action Audio Narration The tubes shown must be moved towards the instrument above one at a time with appearance of the graph. (Please redraw all figures.) The tubes must be moved one at a time towards the instrument which should have a hole in which the tube fits. Every time a tube enters and leaves the hole in the instrument, a part of the graph must simultaneously appear. When tubes 1&2 are analyzed, only a straight line parallel to the x-axis of graph must appear. When the tubes with red dots are analyzed (tube 3), the red hump must appear followed by the violet hump when tube 4 is analyzed and blue hump when tube 5 is analyzed. The fractions are then analyzed for their protein content using a UV-Visible Spectrophotometer at 280 nm and a graph of eluant volume versus protein concentration is plotted. 5

  20. Master Layout (Part 3) 1 Affinity chromatography This animation will consist of 3 sections. Protein purification by: Part 1 – Gel filtration. Part 2 – Ion exchange chromatography Part 3 – Affinity chromatography Mobile phase 2 Proteinmixture Ligand solution 3 Derivatized stationary phase 4 Effluent sample fractions 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

  21. Definitions of the components (Part 3 – Affinity chromatography) 1 1. Derivatized stationary phase: The stationary phase resin in affinity chromatography consists of a covalently bound ligand that will specifically bind the protein of interest by interacting with it. 2. Protein mixture: The unpurified protein mixture consists of proteins having different properties and interaction specificities for the ligand bound to the column matrix. 3. Mobile phase: Following sample loading, the unbound proteins are washed out of the column using a suitable mobile phase. Depending on the protein of interest, this could be either water or sometimes a salt solution. 4. Ligand solution: This solution is passed through the column to elute the bound protein of interest. Since it contains the same ligand that is bound to the column matrix, it is capable of eluting the protein by interacting with it. 5. Effluentsample fractions: The solution leaving the column is collected in suitably sized fractions for further analysis. The unbound proteins are eluted from the column first followed by the bound proteins which are removed after washing with the ligand solution. 2 3 4 5

  22. Part 3, Step 1: 1 2 3 Chromatography column Column packed with derivatized resin Action Description of the action Audio Narration 4 Showing the column being filled with a material. • (Please redraw all figures.) • First show only empty column on the left. • Then the filled column must be shown. The column is packed with a resin that has been covalently coupled to the ligand specific to the protein of interest. 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

  23. Part 3, Step 2: 1 Sample loading 2 Affinity column 3 Unpurified protein mixture Action Description of the action Audio Narration 4 • (Please redraw all figures.) • First show only the container with solution on the left and the column on the right. • Then the container must be lifted and poured into the column as shown on the right. Pouring the solution from the container on the left into the column on the right. The affinity column is loaded with the protein mixture containing various proteins having different properties and interaction specificities. 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

  24. Part 3, Step 3: 1 Mobile phase reservoir Mobile phase 2 Direction of flow 3 Action Description of the action Audio Narration 4 The column is washed with a suitable mobile phase to remove all the unbound proteins. The protein of interest, which has higher affinity for the column, remains bound to the derivatized column matrix and is not removed during the washing. The different shapes must be separated from one another. • (Please redraw all figures.) • Show the column on the left. • The column must be zoomed into and the figure on the right must then be shown. • The squares and the triangles are shown to move down while the red semi-circles are bound to the black dots present on the pink circles. 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

  25. Part 3, Step 4: 1 2 3 Column washing Action Description of the action Audio Narration 4 Liquid coming out of the column must be collected in the tubes. (Please redraw all figures.) Show an empty tube and the column followed by the tube being filled by a plain blue colour. Then the first tube must be moved and a second tube must be filled in a similar way. Then show a third tube being filled with the blue squares and orange triangles. Fractions collected during sample washing can be analyzed, assayed and discarded if they are not required. 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

  26. Part 3, Step 5: 1 Ligand solution 2 3 Column elution Action Description of the action Audio Narration 4 The solution in the container must be poured into the column and sample must be collected. (Please redraw all figures.) Show the column on the left with the solution being poured into it. Then show the sample being collected in the tubes on the right as shown. After the column has been washed thoroughly, the protein of interest is eluted by passing a ligand solution which binds to the matrix-bound protein and removes it from the column. 5 Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

  27. Part 3, Step 6: 1 Desired pure protein Protein impurities Protein concentration 2 UV-Vis spectrophotometer Volume of eluant 3 7 2 1 3 4 5 6 Collected fractions Action Description of the action Audio Narration 4 The tubes shown must be moved towards the instrument above one at a time with appearance of the graph. • (Please redraw all figures.) • Move the tubes one at a time in the numbered order towards the instrument shown to indicate they are being analyzed. • A graph must simultaneously appear as shown on right. When tube 3 is analyzed, blue-orange hump must appear. When tube 5 & 6 are analyzed, the red hump must appear. The fractions are then analyzed for their protein content using a UV-Visible Spectrophotometer at 280 nm. A graph of eluant volume versus protein concentration is then plotted. 5

  28. Interactivity option 1:Step No:1 1 It is desired to separate the peptide hormone insulin (5.8kD) from bovine serum albumin (70kD) and b-lactoglobulin (18.4kD) by gel filtration using a Sephadex G-50 (1.5-30kD) column as shown below. Drag the components from the column into the tubes shown below in the order in which they will elute out from the column. 2 Protein concentration Direction of flow b-lactoglobulin BSA (70 kD) Insulin 3 b-lactoglobulin (18.4 kD) BSA Insulin (5.8 kD) 4 3 2 1 Volume of eluant Results Interacativity Type Options Boundary/limits User should be allowed to drag & drop the 3 groups of coloured circles from the column into the tubes below. The red circles must go in tube-1, the blue circles in tube-2 & the green circles in tube-3. Every time the user drags the circles into the tube correctly, it must be highlighted in green and the corresponding curve in the graph must appear. If he gets it wrong, it must be highlighted in red & the circles must again go back into the column & user can continue. Drag & drop. 5

  29. Interactivity option 2:Step No:1 1 Protein A is a 40-60 kD protein that is found in the cell wall of Staphylococcus aureus. It has very high affinity for the immunoglobulin, IgG and can therefore be purified from a mixture of other proteins by affinity chromatography. Shown below is the impure protein mixture and the steps for the procedure (in the next slide). Place them in the correct order such that pure Protein A can be obtained. 2 Protein A 3 Unpurified protein mixture 4 Results Interacativity Type Options Boundary/limits User must drag & drop the figures given in the next slide into the boxes marked as step 1, 2 & 3. The user must be first shown the figure above with its labels followed by the 3 figures in the next slide. He must be provided with 3 empty boxes marked as step 1, 2 & 3. User has to drag & drop the correct figure into the box. Step 1 is sample loading, step 2 is column washing & step 3 is column elution. Every time the user drags & drops correctly, the box must be highlighted as green. If not, the figure must be removed from the box & user must try again. 5 Drag & drop.

  30. Interactivity option 2:Step No:2 1 IgG 2 Mobile phase 3 Step 2 Step 3 Step 1 IgG solution Column washing Sample loading 4 5 Column elution

  31. Questionnaire 1 1. If a porous gel matrix does not allow proteins above 40kD inside its pores, then what will be the order of elution of four proteins have masses of 12kD, 25kD, 56kD and 80kD? Answers: a) 25kD, 56kD, 12kD, 80kD b) 12kD, 25kD, 56kD, 80kD c) 80kD, 56kD, 25kD, 12kD d)‏ 80kD, 25kD, 56kD, 12kD 2. To elute a strongly negatively charged protein that is bound to an anion exchanger, pH of the buffer should be? Answers: a) pH 2.3 b) pH 3.5 c) pH 6.7 d) pH 9.2 3. Purification by affinity chromtaography relies on Answers: a) Net charge of the protein b) Size of the protein c) Specific interactions with a ligand d) Molecular weight of the protein‏ 4. The ligand for affinity chromatography is attached to the resin by: Answers:a) Covalent bonding b) Van der Waals interactions c) Hydrophobic interactions d) Hydrogen bonding‏ 2 3 4 5

  32. Links for further reading Books: Biochemistry by Stryer et al., 5th & 6th edition Biochemistry by A.L.Lehninger et al., 4th edition Biochemistry by Voet & Voet, 3rd edition

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