Jessica Nguyen Glycoprotein Chromatography Partners: Tyler Maclay, Goldner Figaro

April 26, 2013 BI210X-01: Dr. Knapp

Introduction: Chromatography is a laboratory technique that separates certain components of a mixture from its surroundings through movement of a stationary phase. Chromatography is a useful laboratory technique where it can take advantage of the elements of specificity of certain molecules that are also dependent on charge and active sites as well. Ligands are sugar molecules that have a tendency to bind to specific active sites on certain molecules in a cell. The binding of any ligand may have several purposes such as serving a biological purpose to signal triggering molecular binding sites on a target protein (Baron 2010). This interaction of the binding of a protein and a ligand can be manipulated to serve as a purification process. In serum, which is centrifugation of proteins from blood, proteins that have certain ligand specificity will bind to only one specific ligand. Therefore, the manipulation of this binding between protein-ligand complexes can be taken advantage of through means of affinity chromatography. The objective of chromatography is to separate elements. Thin layer chromatography is a separation technique that is commonly used in chemistry while affinity chromatography is commonly used in biological experimentation. In terms, of chemistry, thin layer chromatography is one of the more preferred methods of separation due to the effectiveness and credibility for the natural mixtures. There are different mixtures that travels and move at different paces depending on what solution is being use as the adsorbent and as well as other different partitions. The components of the mixtures also have an effect on how fast or slow the mixture will travel. Separation through the means of chromatography be may preparative or analytical. The whole purpose of preparative chromatography is to separate several components of a mixture into larger quantities with large similarities. On the other hand, analytical chromatography is normally associated with smaller quantities of materials to determine relative proportions of unknowns in a mixture. There are several purposes of thin layer chromatography that include the purposes to establish the number of components in a mixture of organic compounds, to establish the identity of an unknown substance, to monitor the progress of an organic reaction, to determine the effectiveness of a purification, to determine the appropriate solvent system for column chromatography, and to monitor the column chromatographic separation of organic compounds by testing fractions. Due to thin layer chromatography being a reliable technique for separation, it is usually the preferred method of isolation of a substance or several unknown purified substances for column chromatography and gas chromatography which typically follow after. An important factor of thin layer chromatography is the solvent as well as the adsorbent. Silica gel, cellulose, and alumina are usually the coated surfaces of chromatography plates. They are the adsorbents on which the actual separation is carried out on the thin layer. The important factor of these adsorbents to be reliable separators is when the plate is actually placed in the solvent system, the solvent front will move and as it moves different compounds move to different extents on the surface of the plate due to continuous differential absorption and differential elution occurring between the stationary and mobile solvent phase. The polarity of the solvent system is one of the many important factors that determine the effectiveness of TLC as an analytical tool.

Thin layer chromatography can be used as an analogy for affinity chromatography. The ideal mechanism of affinity chromatography follows the binding and charge nature of thin layer chromatography. Affinity chromatography is more effective for biological experimentation due to the difference of substances that are tested. This method of chromatography is extremely successful where it allows examiners to be able to isolate certain large molecules such as proteins from a selection of mixtures of proteins with little margin for error. The concept of the solvent phase for affinity chromatography is the idea behind certain molecule binding beads that are used to block certain solvents. Affinity chromatography follows the mechanism that any substance can be isolated by running a binding mobile structure for ligands to bind. Affinity chromatography is a versatile method that is extremely effective and is usually used in the process of the purification of a protein-ligand binding complex. The separation of a few milligrams to a hundred grams can be done efficiently. The efficiency of affinity chromatography is dependent on the sampling moving down a column continuously partitioning occurs between the moving solvent from the stationary adsorbent, which in this case are concanavalin-A beads. The protein sample that is to be separated is applied at the top of the affinity column, where it begins to journey downwards through a column in a mobile phase. Similar to TLC, the moving solvent is visibly moving through the column and depending on the binding affinity of the beads that are being used as the adsorbed on the surface of the adsorbent to a different extent. The suitable solvent with similar soluble characteristics in this case mannose, can be used to extract the bound protein from the binding sites of the concanavalin beads as an elutant that will allow the separation at an effective and constant rate. One aspect of affinity chromatography that is essential is the subjection of samples through electrophoresis. The gel that is used during the subjection to electrophoresis is SDS. SDS is used as a denaturation agent that masks the native charges around any proteins, and provides all proteins with a negative charge. During the analysis of samples that have been subjected to electrophoresis, the samples are purely traveling based on their molecular weights. Due to the general process of running electrophoresis and creating an electric field for the proteins to travel to a positive end, which results in the heavier protein complexes to travel slower covering less distance on the gel. This allows the analysis and determination of which protein has been isolated. Electrophoresis subjection of isolated samples are often run alongside sample proteins that serve as a control on a standard curve. With proteins and a standard curve of molecular weights, the unknown protein can be identified through means of a semilog graph that indicates the relationship of proteins through molecular weights and the distance traveled after electrophoretic subjection. Using the principles of chromatographic separation, affinity chromatography was used to analyze the objective of the experiment, which was to determine which glycoproteins of red blood cells contain glucose/mannose. Con-A bound to agarose beads run a mixture of glycoproteins to see which contains glucose or mannose, which will bind to Con-A, then run the flow through and elutant through SDS-chromatography against standard proteins. Their relative weights can then be used to identify which proteins contain mannose or glucose and which proteins dont. Methods: Reagents: Con-A bound affinity column Standard Proteins SDS-agarose gel 0.2M MgCl

Mannose (Elutant) Commasie Blue Stain Electrophoresis buffer, 2x concentration Acetic Acid Extraction buffer (0.05 M Tris-HCl pH 7.3) Column buffer (0.15M NaCl, 10 mM Tris pH 8.0) Research Design: Modified from Poole and Hancock. l984. Eur.J.Biochem.144:607-612 I. Preparation of sample 1. Obtained 2 plastic sample test tubes and filled each with half of dog blood sample. 2. Subjected two test tubes with dog blood samples for centrifugation for 10 minutes at 5000 rad/s to separate blood pellets from sample. II. Osmotic shock of sample 1. Resuspended blood pellets, both test tubes, from centrifugation in 8.0mL of 0.05M Tris-HCl pH 7.3 and 0.2M MgCl. 2. Incubated for 10 minutes. 3. Chilled samples in a cold ice-water bath for 15 minutes. 4. Warmed at 30C for 10 minutes. 5. Subjected RBC contents to further centrifugation to pellet out RBC contents. III. Application of samples to column w/mannose elution 1. Run supernatant samples through ConA beaded column. 2. Collect flow through fraction. 3. Wash column with column buffer twice. 4. Elute with mannose. 5. Collect eluted fraction. 6. Prepare 20 L flow through fraction w/electrophoresis buffer (20 mL) Prepare 20 of eluted fraction w/electrophoresis buffer (20 L) 7. Run prepared samples with standard proteins. IV. Application of samples to column w/SDS elution buffer 1. Run supernatant samples through ConA beaded column. 2. Collect flow through fraction. 3. Wash column with column buffer twice. 4. Elute with affinity chromatography SDS elution buffer 5. Collect eluted fraction. 6. Prepare 20 L flow through fraction w/electrophoresis buffer (20 mL) Prepare 20 of eluted fraction w/electrophoresis buffer (20 L) 7. Run prepared samples with standard proteins.

Results: Figure #1: SDS Electrophoresis Gel

Table #1: SDS Gel Key Well 1 2 3 4 5 6 Sample Standard Proteins Supernatant Flow Through Fraction (Mannose Elutant) Eluted Fraction (Mannose Elutant) Flow Through Fraction (SDS Elution Buffer) Eluted Fraction (SDS Elution Buffer)

Discussion: The standard proteins run different bands along the scale from the electrophoresis subjection. SDS PAGE gel is responsible for masking the native charges of proteins. These charges are all given an overall negative charge. The nature of electrophoresis requires that contents of the wells are subjected to a positive electrical field. This promotes the movement of proteins purely through molecular weight. The heavier a protein is the more likely it is to be located near the well origin. In this lab experiment, there were two trials to elute the proteins that had a binding affinity to concanavalin A beads that were used as a mobile phasing agent in the column. The two variations between the trials was simply during the elution of the bound protein process. One trial of elute was done using mannose which binds to concanavalin A and has a high affinity. The mechanism behind this binding was the reasoning that the proteins that actually bound to the con-A beads would be eluted while mannose would bind to the con-A beads. Standard SDS elation buffer was used to elute proteins that bound to com-A. These two methods are used in comparison to one another to determine whether glycoproteins in blood serum could be effectively isolated through means of affinity chromatography with con-A beads. This lab required the centrifugation of dog blood samples to smaller pellets. The centrifugation process separated the plasma layer from the blood pellet layer. The pellets were too big to even put through the columns, therefore they required further centrifugal ion as well as shocking the red blood cell pellets to allow for the exploitation of the glycoproteins without

completely denaturing the proteins themselves. Pellets of blood samples with proteins were shocked with a high concentration of MgCl to expose the proteins from the normal nature. From figure #1 where well 3 and well 4 indicate the flow through fractions of both the SDS elution buffer and that from the mannose elution process; the results show visible signs of proteins traveling several different distances starting from the origination of the wells. The flow through fractions contained more proteins than that in the eluted fraction because there. Only supposed to be a single one or two glycoproteins that could actually bind to the con-A beads. From wells #4 and #6, one can see that there are no protein bands. This indicates that no proteins that were bound to the com-A beads were successfully eluted from the column. Although there are no proteins in the eluted fraction on the gel, the flow through fractions of the gel present indicate that there were proteins that successfully migrated through the column. This shows that there may not have been a high binding affinity to the proteins. Although there were proteins that have a binding nature to con-A beads, the column buffer may have had a stronger push to pull the beads through the flow through fraction which would leave nothing for the elution fraction afterwards. The supernatant was run along the samples of flow through fraction of both trials as a control for what proteins were in the dog blood samples. During the elution processes of the experiment, the results did not support the hypothesis as an indicated result that the procedure would
successfully isolate the proteins that supposedly bound to the con-A beads. This may have been a factor of a few sources of error, such as running the supernatant through the column, osmotic shocking of the the red blood cell samples as well as the preparing of the samples for electrophoresis subjection. While the samples were introduced to the column, there were several washed to prevent any of the supernatant and the impurities of the samples. During the washes, the column had a separated layer and just the beaded layer. The elution buffer was added in both situations of mannose elute not and SDS elution buffer, there was a small ring of red that traveled just below the line of the conjugated con-A beads. This can possibly be concluded as the proteins that were bound to the beads. Because the red blood cell pellets are too big even after centrifugation, to travel through the column. Due to the large nature of the pellets, it was not possible for the pellets to actually pass through the con-A beads that were so tightly packed together. From the SDS gel selection, one could see that there were no protein bands in either of the eluted fraction wells which shows that no proteins were successfully eluted during the process. One research aspect that this sparked was how one can efficiently isolate proteins using chromatography techniques. Another question that was directly formulated from the procedure proposed in the experiment is how can one successfully pellet the red blood cell pellets to be small enough to go through the con-A columns.