Protein separation techniques Chromatography Purified proteins are essential

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. Harini Chandra Affiliations

1 Master Layout Chromatography Gel filtration Ion exchange chromatography Affinity chromatography 2 3 4 5 Action The three images must appear with each of them being a separate tab. Description of the action 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. Audio Narration 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. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook); Biochemistry by Voet & Voet (3 rd edition)

1 Definitions of the components: (Master layout) 2 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. 3 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. 4 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. 5

1 Master Layout (Part 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 (3 rd edition)

1 2 Definitions of the components (Part 1 – Gel filtration) 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 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 4. Effluent sample 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. 5

1 2 3 4 5 Analogy 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”.

1 Part 1, Step 1: 2 Size exclusion gel matrix 3 4 Chromatography column Action Showing the column being filled with a material. Packed gel column Description of the action (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. 5 Source: Biochemistry by Voet & Voet (3 rd edition) Audio Narration The column is packed with a hydrated porous gel matrix suitable for the required protein separation. •

Part 1, Step 2: 1 Sample loading 2 Packed gel column 3 Porous gel matrix Unpurified protein mixture 4 5 Action Pouring the solution from the container on the left into the column on the right. Description of the action (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. Source: Biochemistry by Voet & Voet (3 rd edition) Audio Narration 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.

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

Part 1, Step 4: 1 2 3 4 5 Sample collection 1 Action Liquid coming out of the column must be collected in the tubes. 1 2 3 1 2 Description of the action (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. Source: Biochemistry by Voet & Voet (3 rd edition) 3 4 5 Audio Narration Fractions of appropriate size are collected 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.

1 Part 1, Step 5: 2 3 1 2 3 4 5 Collected fractions 4 5 Action The tubes shown must be moved towards the instrument above one at a time with appearance of the graph. Protein concentration UV-Vis spectrophotometer Highest molecular weight Lowest molecular weight Volume of eluant Description of the action (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. Source: Biochemistry by Voet & Voet (3 rd edition) Audio Narration Each fraction is analyzed for its protein content using a UVVisible Spectrophotometer at 280 nm. A graph of eluant volume versus protein concentration is then plotted.

1 Master Layout (Part 2) 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 3 4 5 Protein mixture Charged stationary phase Effluent sample fractions Source: Biochemistry by Voet & Voet (3 rd edition) High salt elution buffer

1 2 Definitions of the components (Part 2 – Ion exchange chromatography) 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 (DEAEcellulose), which is an anion exchanger. 3 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. 4 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 p. H 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. 5 4. Effluent sample 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.

1 Part 2, Step 1: 2 3 Chromatography column 4 5 Action Showing the column being filled with a material. Packed ion exchange column Description of the action (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. Source: Biochemistry by Voet & Voet (3 rd edition) Audio Narration 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.

1 Part 2, Step 2: + + Net negative charge + +-+ -+++- + - Net positive charge Large net positive charge 2 Packed ion exchange column 3 Sample loading Protein sample loaded + - ++ -++ +- +Unpurified protein mixture 4 5 Action Description of the action Pouring the solution from the container on the left into the column on the right. (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. Source: Biochemistry by Voet & Voet (3 rd edition) Audio Narration 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.

1 Part 2, Step 3: Mobile phase reservoir 2 Low salt elution buffer - -- 3 4 5 +++ ++ ++ + + + + - -Action Description of the action 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. Source: Biochemistry by Voet & Voet (3 rd edition) + + + + Direction of flow - -- Audio Narration The column is first eluted with a buffer solution having low salt concentration or a suitable p. H such that the negatively charged molecules are removed from the column while the positively charged molecules remain bound to the anion exchange resin.

1 2 Part 2, Step 4: High salt elution buffer 3 4 5 Mobile phase reservoir Direction of flow + Action The blue coloured circles must now be moved downward. Description of the action (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. • • Source: Biochemistry by Voet & Voet (3 rd edition) + + Audio Narration The buffer solution is then changed to one having a high salt concentration or such that the net p. H 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.

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

2 UV-Vis spectrophotometer 3 Protein concentration Part 2, Step 6: 1 Large negative charge Large positive charge Volume of eluant 1 2 -3 ++ ++ ++ + 4 5 Collected fractions 4 5 Action The tubes shown must be moved towards the instrument above one at a time with appearance of the graph. Description of the action (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. Audio Narration 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.

1 2 Master Layout (Part 3) 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 Protein mixture 3 4 5 Ligand solution Derivatized stationary phase Effluent sample fractions Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

1 2 Definitions of the components (Part 3 – Affinity chromatography) 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 4 5 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. Effluent sample 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.

1 Part 3, Step 1: 2 3 4 Chromatography column Action Showing the column being filled with a material. Column packed with derivatized resin Description of the action (Please redraw all figures. ) • First show only empty column on the left. • Then the filled column must be shown. • Audio Narration 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, 4 th edition (ebook)

1 Part 3, Step 2: Sample loading 2 Affinity column 3 Unpurified protein mixture 4 5 Action Pouring the solution from the container on the left into the column on the right. Description of the action (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. • Audio Narration The affinity column is loaded with the protein mixture containing various proteins having different properties and interaction specificities. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

1 Part 3, Step 3: Mobile phase reservoir Mobile phase 2 Direction of flow 3 4 5 Action The different shapes must be separated from one another. Description of the action (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. • • Audio Narration 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. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

Part 3, Step 4: 1 2 3 4 5 Column washing Action Liquid coming out of the column must be collected in the tubes. Description of the action (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. Audio Narration Fractions collected during sample washing can be analyzed, assayed and discarded if they are not required. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

Part 3, Step 5: 1 2 Ligand solution 3 4 5 Column elution Action The solution in the container must be poured into the column and sample must be collected. Description of the action (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. Audio Narration After the column has been washed thoroughly, the protein of interest is eluted by passing a ligand solution which binds to the matrixbound protein and removes it from the column. Source: Biochemistry by A. L. Lehninger, 4 th edition (ebook)

Part 3, Step 6: 1 Protein impurities Desired pure protein Protein concentration 2 UV-Vis spectrophotometer Volume of eluant 3 1 4 5 2 3 Action The tubes shown must be moved towards the instrument above one at a time with appearance of the graph. 4 7 5 6 Collected fractions Description of the action (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. • • Audio Narration 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.

1 Interactivity option 1: Step No: 1 It is desired to separate the peptide hormone insulin (5. 8 k. D) from bovine serum albumin (70 k. D) and b-lactoglobulin (18. 4 k. D) by gel filtration using a Sephadex G-50 (1. 5 -30 k. D) 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. BSA (70 k. D) b-lactoglobulin (18. 4 k. D) 2 Direction of flow 3 4 1 2 5 b-lactoglobulin Insulin BSA Volume of eluant 3 Interacativity Type Drag & drop. Protein concentration Insulin (5. 8 k. D) Options User should be allowed to drag & drop the 3 groups of coloured circles from the column into the tubes below. Boundary/limits Results 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.

1 2 Interactivity option 2: Step No: 1 Protein A is a 40 -60 k. D protein that is found in the cell wall of Staphylococcus aureus. It has very high affinity for the immunoglobulin, Ig. G 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. Protein A 3 Unpurified protein mixture 4 Interacativity Type 5 Drag & drop. Options User must drag & drop the figures given in the next slide into the boxes marked as step 1, 2 & 3. Boundary/limits Results 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.

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

1 Questionnaire 1. If a porous gel matrix does not allow proteins above 40 k. D inside its pores, then what will be the order of elution of four proteins have masses of 12 k. D, 25 k. D, 56 k. D and 80 k. D? 2 3 Answers: a) 25 k. D, 56 k. D, 12 k. D, 80 k. D b) 12 k. D, 25 k. D, 56 k. D, 80 k. D c) 80 k. D, 56 k. D, 25 k. D, 12 k. D d) 80 k. D, 25 k. D, 56 k. D, 12 k. D 2. To elute a strongly negatively charged protein that is bound to an anion exchanger, p. H of the buffer should be? Answers: a) p. H 2. 3 b) p. H 3. 5 c) p. H 6. 7 d) p. H 9. 2 3. Purification by affinity chromtaography relies on 4 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 5

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