Using Ion Exchange Chromatography to Separate Proteins
This activity was developed in collaboration with David Peers, Genentech, Inc.
Students will explore protein purification by separating a positively charged molecule, lysozyme, from a negatively charged molecule, albumin, at neutral pH using ion exchange chromatography.
INFORMATION FOR THE TEACHER
Since 75% of the dry matter in living things is protein, biologists often are trying to purify a protein of interest from other proteins in a cell. To separate any of the macromolecules, scientists utilize their knowledge of the chemistry of these molecules, including: the molecular weight of the protein, its charge, and its shape.
Chromatography is used to separate organic compounds on the basis of their charge, size, shape, and their solubilities. A chromatography consists of a mobile phase (solvent and the molecules to be separated) and a stationary phase either of paper (in paper chromatography) or glass beads, called resin, (in column chromatography) through which the mobile phase travels. Molecules travel through the stationary phase at different rates because of their chemistry.
In gel filtration chromatography, microscopic glass beads with even tinier holes are packed into a column. A sample in a solvent is passed through the beads. Molecules that are smaller than the holes in the beads get hung up in the beads. Therefore, the smaller molecules move through the column more slowly than larger molecules. Depending on the molecules, proteins may be separated, based on their size alone, and fractions containing the isolated proteins can be collected.
In affinity chromatography, a molecule (usually an antibody) that will bind to the protein to be purified is attached to the glass beads. A mixture of proteins is added to the column and everything passes through except the protein of interest which binds to the antibody. Another buffer is used to get the protein to elute from the column. Often this elution buffer contains high concentrations of salt or acid.
In ion exchange chromatography, the glass beads of the column have a charge on them (either + or -). A mixture of proteins is added to the column and everything passes through except the protein of interest. This is because the charge of the beads is picked to have the opposite charge of the protein of interest. If the charge on the bead is positive, it will bind negatively charged molecules. This technique is called anion exchange. If the beads are negatively charged, they bind positively charged molecules (cation exchange). Thus, a scientist picks the resin to used based on the properties of the protein of interest. During the chromatography, the protein binds to the oppositely charged beads. Once the contaminant protein is separated from the protein of interest, a high salt buffer is used to get the desired protein to elute from the column.
INSTRUCTIONS FOR THE STUDENT
Can lysozyme (+ charge at pH 7.2) be separated from albumin (- charge at pH 7.2) by ion exchange chromatography?
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- Using a pipette, add 2 ml. of suspended DEAE SEPHAROSE (anion) resin to a chromatographic column. Let it settle by gravity until you see a very defined, settled top.
- Take off the stopper and let the buffer drip through until there remains only a tiny bit on top of the column. Restopper the bottom.
- Add another 10 ml. of equilibration buffer and let it pass through the column. Stopper it before the column runs completely dry.
- Number 3 sets of test tubes 1-6.
- Gently add .2 ml. of the appropriate sample (without disturbing the top of the bed): Either lysozyme, albumin, or lysozyme + albumin (add .4 ml if you are in this group) to the top of the appropriate column. Unstopper the column and allow the sample to "load" into the column. Collect the flow through into tube #1. Stopper it just before the column runs dry.
- Add another 2 ml. of equilibration buffer and let it pass through each column. Collect the flow through into test tube #2.
- Repeat step 6 three more times collecting fractions into test tubes #3-5. Stopper the column just before the column runs dry.
- While the column is "running", step up test tubes for the protein assay. Number test tubes 1A-F for the lysozyme column, 2A-F for the albumin column, and 3A-F for the combined protein column. Into each test tube place 5 ml of BIORAD reagent.
- Add 2 ml. of elution (high salt) buffer to the top of the column. Unstopper it.
- As the elution comes through collect the 2 ml. sample in test tube #6.
- Add 10 drops of sample collected from each column to the appropriate A-F tube.
- Add another 2 ml. of equilibration buffer to the columns and let it pass through the columns to clean them.
- Determine the absorbance at 595 nm. of each fraction.
- Plot the absorbance of the fractions (line graph) at 595 nm for the fractions from the combined protein chromatograph.
QUESTIONS FOR STUDENTS
- What evidence do you have that the ion exchange column separated the two proteins? Describe what is happening inside the column(s).
- What information have you gained about the characteristics of lysozyme and albumin?
- What results might you have expected if you used a resin with an opposite charge?
- Give some practical applications for column chromatography.