Affinity Chromatography and SDS-PAGE

Introduction Often one of the goals in biochemistry and molecular biology is the isolation of a single pure protein from a complex mixture of proteins. Most fractionation procedures involve many separations. To monitor such protein isolation, electrophoresis is the method of choice. Even though electrophoresis can be considered a type of fractionation in its own right, another popular technique is chromatography. Here, proteins are separated from each other as they are passed through and interact with a matrix. No electric field is used here, but the fractionation procedure relies on the flow of the solvent to aid in separation. There are many types of chromatographic matrices available commercially; each one is designed to exploit the physical and chemical differences that exist among proteins. In gel filtration chromatography, matrix beads of different sizes are used to separate proteins based upon molecular size. With this technique, larger proteins pass readily between beads whereas smaller proteins are retarded in their passage through the matrix as they pass through holes in the beads. Gel filtration chromatography is most useful when attempting to separate proteins that differ greatly in size. In ion-exchange chromatography, differences in protein native charge are used as the basis of separation. Proteins that are positively charged at a particular pH will be attracted to negatively charged matrix beads, whereas the negatively charged beads will repel negatively charged proteins. The reverse situation is also true using positively charged beads. However, one of the main disadvantages of both of these chromatographic procedures is that neither technique will isolate a single protein. For example, gel filtration separates proteins that are similar in size from those that are dissimilar. With this technique, it is not really possible to separate those proteins that are similar in size. Ion exchange chromatography has the same limitations since proteins that have similar charges cannot be separated from each other. Another chromatographic technique called affinity chromatography is capable of separating a single protein from a complex mixture of proteins. It does so because it takes advantage of the interaction of one molecule with a second molecule (ligand). Many types of molecules can be separated using affinity chromatography. The following table lists just a few of these molecules.

Substance isolated by Affinity Chromatography Enzyme Antibody Polysaccharide, glycoprotein Nucleic acid binding protein Hormone receptor

Ligand Substrate or Cofactor Antigen, Virus, Cell Lectin Nucleic acid Hormone

The basic procedure for affinity chromatography is outlined in Figure 1. The first step involves the covalent binding of the complementary binding molecule or ligand to the matrix, usually small agarose beads. The matrix is then poured into a column and allowed to settle. The column is then washed several times to remove any impurities and to equilibrate the column with the appropriate buffer. The mixture of proteins is then applied to the column and allowed to interact with the matrix beads as the solution moves through the column by gravity. During this time, the protein to be isolated will bind specifically to the ligand on the beads while the remaining proteins will pass through the column. The column is then washed with buffer to remove any proteins that are not specifically bound to the column. Finally, the protein bound to the column is then eluted from the column under

specific conditions that will disrupt the interaction of the protein with the ligand. The solution containing the protein is collected in a tube as it drips from the column. The final step involves some type of assessment of the chromatography to make sure that the protein is pure. As mentioned previously, electrophoresis is often used. In this experiment, you will be isolating horse serum albumin from diluted horse serum by affinity chromatography. The matrix in the column will be Affi-Gel blue, which contains a reactive blue dye molecule (Cibacron Blue F3GA) covalently linked to agarose beads. To elute the albumin from the column, a 1% solution of SDS will be used. You will then assess the chromatographic run by subjecting your sample of albumin, the column flowthrough material (all other serum proteins), and the diluted horse serum to SDS-PAGE following by staining with Coomassie blue. electrophoresed with your samples: The following standard proteins will be

Standard Protein Bovine serum albumin, dimer Bovine serum albumin, monomer Ovalbumin Myoglobin

Molecular Weight (in Daltons) 132,000 66,000 43,000 17,000

Procedure: Preparation of the Affi-Gel Affinity Column 1. Secure your column in a vertical position on a ring stand such that the narrow, plugged end of the column is facing downward. The porous disc in the bottom of the column ensures that the matrix beads stay in the column, but allows solutions and proteins to flow freely through the column. 2. Place a small beaker under the column to catch the buffer and any flow-through. Since the Affi-Gel blue is supplied as a slurry of agarose beads in a buffer, mix the slurry thoroughly to make sure that the solution is suspended uniformly. Quickly transfer 2 mL of the slurry to your column. As the Affi-Gel blue is settling, you will notice two layers forming: a bottom layer of Affi-Gel blue and a top layer of buffer. Allow this to settle for several minutes. 3. Carefully remove the blue plug at the bottom of the column. Buffer should begin to flow dropwise into the beaker below. When the level of the buffer approaches the top of the Affi-Gel blue layer, fill the column with column buffer (0.15 M NaCl, 10 mM Tris, pH 8.0) and allow the solution to flow through the column. Repeat this step. NEVER ALLOW THE AFFI-GEL TO BECOME DRY. If necessary, add more column buffer to prevent it from becoming dry. Applying the Horse Serum 1. Obtain five small microcentrifuge tubes and label tubes #1, #2, #3, flow through, and bound. Pipet 10 L of the diluted horse serum in tube #1. 2. After the column has been washed twice, allow the buffer to drain to within 1 or 2 mm of the top of the Affi-Gel blue. Carefully pipet 500 L of the horse serum into the column and immediately place the tube marked flow through under the column and collect the solution that contains the horse serum minus those proteins bound to the column. 3. When the sample has been collected, remove the tube flow through and wash the column twice with column buffer. 4. Allow the final wash to drain to within several mm of the top of the matrix. Pipet 1 mL of elution buffer (1% SDS) into the column and immediately place the tube marked bound under the column and collect the solution that contains the protein bound to the column.

Sample Preparation for SDS-PAGE 1. Add 10 L of distilled water to the 10 L serum sample in tube #1. 2. Pipet 20 L of the contents of the tube labeled flow through to tube #2. 3. Pipet 20 L of the contents of the tube labeled bound into tube #3. 4. Add 20 L of SDS-PAGE sample buffer to each of the tubes labeled #1, #2, and #3. Mix thoroughly. Boil tubes for 5 minutes in a boiling water bath. 5. Load 10 L of each of the samples (#1-#3) into separate lanes of an SDS-PAGE gel. Also load 10 L of the standard proteins in an adjacent well. 6. Electrophorese the samples and stain the gels overnight in Coomassie blue as detailed in the previous laboratory exercise. 7. Obtain a digital picture of your stained gel for your lab report. Label each lane plus all four standards with molecular weights, and title your drawing Figure 1. SDS-PAGE of affinity chromatography procedure used to isolate albumin from horse serum. Questions to Answer: 1. Describe the results of your albumin purification including a description of the relative mobility of the purified protein. Did your affinity chromatography column remove all of the albumin from the serum? How do you know? How pure was the affinity-isolated protein? 2. An antiserum contains hundreds of proteins in addition to antibodies and is therefore a complex, impure mixture of proteins. You have been asked by the Centers for Disease Control and Prevention to test people to determine if they have been exposed to the bovine spongiform encephalopathy virus that causes mad cow disease. Design an affinity chromatography experiment to purify antibodies that react with thevirus from serum you have collected from these people. Be specific in your methodology. Would either influenza virus or respiratory syncycial virus (RSV) serve as a good ligand in this experiment? Why? 3. In the chromatography experiment you performed in lab, the serum albumin was eluted from the column using 1% SDS. albumin-ligand interaction? What, specifically, did this detergent do to the If you were interested in isolating albumin in its native

conformation, would you elute the albumin from the column with SDS? If not, what

other method could you use to elute the albumin in this experiment and still preserve its structure? What to turn in: Figure 1, questions and answers to questions

1. Couple ligand to matrix covalently.

3. Elute pure protein

+
Matrix beads

elution buffer
ligand
Purified protein

2. Apply sample containing protein plus impurities and wash away impurities.

Figure 1. General principles of affinity chromatography. Protein (antibody ) impurities