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Chromatography

INTRODUCTION

IN THIS LAB, WE WILL BE EXPRESSING THE GREEN FLUORESCENT PROTEIN (GFP) IN BACTERIA AND PURIFYING IT USING COLUMN CHROMATOGRAPHY. THE SPECIFIC TYPE OF CHROMATOGRAPHY WE WILL BE USING IS HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC). THE FOLLOWING IS INFORMATION FROM BIO-RAD INC., THE SUPPLIER OF THE REAGENTS:

GFP has several stretches of hydrophobic amino acids, which results in the total protein being very hydrophobic. When the supernatant, rich in GFP, is passed over a HIC column in a highly salty buffer (Binding Buffer), the hydrophobic regions of the GFP stick to the HIC beads. Other proteins which are less hydrophobic (or more hydrophilic) pass right through the column. This single procedure allows the purification of GFP from a complex mixture of bacterial proteins.

Loading the GFP supernatant onto the chromatography column When students load the GFP supernatant onto their columns, it is very important that they do not disturb the upper surface of the column bed when performing the chromatography procedure. The column matrix should have a relatively flat upper surface. A slightly uneven column bed will not drastically affect the procedure. However, subsequent steps of loading, washing, and eluting should minimize disrupting the column such that beads "fluff up" into the buffer. When loading the GFP supernatant onto the column, the pipette tip should be inserted into the column and should rest against the side of the column. The supernatant should be slowly expelled from the pipette, down the walls of the column. When the supernatant has completely entered the column, a green ring of fluorescence should be visible at the top of the bed when viewed with the UV light. There are four different buffers which are used in the HIC procedure. Each buffer should be slowly pipetted down the side of the column to minimize disturbance to the column resins.

Equilibration buffer A medium salt buffer (2 M (NH4)2SO4) which is used to "equilibrate" or "prime" the chromatography column for the binding of GFP.

Binding buffer An equal volume of high salt Binding Buffer (4 M (NH4)2SO4) is added to the bacterial lysate. The end result is that the supernatant containing GFP has the same salt concentration as the equilibrated column. When in a high salt solution, the hydrophobic regions of proteins are more exposed and are able to interact with and bind the hydrophobic regions of the column.

Wash buffer A medium salt Wash Buffer (1.3 M (NH4)2SO4) is used to wash weakly associated proteins from the column; proteins which are strongly hydrophobic (GFP) remain bound to the column. When the Wash Buffer is applied to the column, care should again be taken to minimize disturbance of the column resin. Slow, steady pipetting down the side of the column is most effective. At this point "a ring" of GFP should begin to penetrate the upper surface of the matrix (~ 1–2 mm into the bed).

Elution buffer A low salt buffer (TE Solution; 10 mM Tris/EDTA) is used to wash GFP from the column. In low salt buffers (which have a higher concentration of water molecules), the conformation of GFP changes so that the hydrophilic residues of GFP are more exposed to the surface, causing the GFP to have a higher affinity for the buffer than for the column, thereby allowing the GFP to wash off the column. The 0.75 milliliters of TE (Elution) Buffer is applied to the column gently, as described above. The TE Solution will disrupt the hydrophobic interactions between the GFP and the column bed, causing GFP to let go and "elute" from the column. The GFP should pass down the column as a bright green fluorescent ring. This is easily observed using the UV light. If the column bed was disturbed in any of the preceding steps, the GFP will not elute as a distinct ring, but will elute with a more irregular, distorted shape. However, elution should still occur at this step. If successful, collection tube 3 should fluoresce bright green.

Day 1: Lysis for Chromatography

MATERIALS

CULTURE OF JM109/PGFP Microtubes Pipette Microtube rack Marker Beaker of water for rinsing pipettes TE solution Lysozyme (rehydrated)

Procedure

1. TRANSFER BACTERIAL CULTURE INTO A MICROFUGE TUBE AND SPIN FOR 5 MINUTES IN THE CENTRIFUGE AT MAXIMUM SPEED. BE SURE TO BALANCE THE TUBES IN THE MACHINE.
2. Pour off the liquid supernatant above the pellet. After the supernatant has been discarded, there should be a large bacterial pellet remaining in the tube. You may observe the pellet under UV light to check GFP abundance.
3. Add 250 µl of TE Solution to each tube. Resuspend the bacterial pellet thoroughly by carefully pipetting up and down several times with the pipette.
4. Using the P1000 micropipette, add 1 drop of lysozyme to the resuspended bacterial pellet. Cap and mix the contents by flicking the tube with your index finger. The lysozyme will start digesting the bacterial cell wall. Observe the tube under the UV light. Place the microtube in the freezer until the next laboratory period. The freezing will cause the bacteria to explode and rupture completely.

Day 2: Chromatography

MATERIALS

MICROTUBES Beaker of water for rinsing pipettes HIC chromatography column Column end cap Waste beaker or tube Binding Buffer Equilibration Buffer Collection tubes Wash Buffer Equilibration Buffer TE Buffer UV light

Procedure

1. REMOVE YOUR MICROTUBE FROM THE FREEZER AND THAW IT OUT USING HAND WARMTH. FINGER VORTEX THOROUGHLY TO SOLUBILIZE GFP. PLACE THE TUBE IN THE CENTRIFUGE AND PELLET THE INSOLUBLE BACTERIAL DEBRIS BY SPINNING FOR 10 MINUTES AT MAXIMUM SPEED. LABEL A NEW MICROTUBE WITH YOUR TEAM’S INITIALS.
2. While you are waiting for the centrifuge, prepare the chromatography column. Before performing the chromatography, shake the column vigorously to resuspend the beads. Then shake the column down one final time, like a thermometer, to bring the beads to the bottom. Tapping the column on the table-top will also help settle the beads at the bottom. Remove the top cap and snap off the tab bottom of the chromatography column. Allow all of the liquid buffer to drain from the column (this will take ~3–5 minutes).
3. Prepare the column by adding 2 ml of Equilibration Buffer to the top of the column, 1 ml at a time using a well rinsed pipette. Drain the buffer from the column until it reaches the top of the white column bed, around the 1 ml mark. Cap the top and bottom of the column.
4. After the 10 minute centrifugation, immediately remove the microtube from the centrifuge.
5. Examine the tube with the UV light. The bacterial debris should be visible as a pellet at the bottom of the tube. Note the color of the pellet and the supernatant. Using a new tip, transfer 250 µl of the supernatant into the new microtube. Again, rinse the pipette well for the rest of the steps of this lab period.
6. Using a new tip, pipette250 µl of Binding Buffer to the microtube containing the supernatant.
7. Obtain 3 collection tubes and label them 1, 2, and 3. Place the tubes in a rack. Remove the cap from the top and bottom of the column and let it drain completely into a liquid waste container. When the last of the buffer has reached the surface of the HIC column bed, gently place the column on collection tube 1. Do not force the column tightly into the collection tubes—the column will not drip.
8. Using a new tip, carefully load 250 µl of the supernatant (in Binding Buffer) into the top of the column by resting the pipette tip against the side of the column and letting the supernatant drip down the side of the column wall. Let the entire volume of supernatant flow into tube 1. Examine the column using the UV light. Note your observations in the data table below.
9. Transfer the column to collection tube 2. Using the rinsed pipette and the same loading technique described above, add 250 µl of Wash Buffer and let the entire volume flow into the column. As you wait, predict the results you might see with this buffer. Examine the column using the UV light and list your results in the data table below.
10. Transfer the column to tube 3. Using new tip, add 750 µl of TE buffer (Elution Buffer) and let the entire volume flow into the column. Again, make a prediction and then examine the column using the UV light. List the results in the data table.
|Chromatography Data Table |
| |Observations (color) |
|Tube 1 | |
|Tube 2 | |
|Tube 3 | |

11. Examine all of the collection tubes using the UV lamp and note any differences in color between the tubes.

QUESTIONS FOR YOUR REPORT (10 PTS)

PRESENT A ½ PAGE SUMMARY OF PURPOSE OF THIS LAB, WHAT METHODS YOU USED, AND YOUR RESULTS/CONCLUSIONS.

Explain the basic principle of HIC.

In preparing cells for chromatography, explain the importance of adding lysozyme and the freezing step.

Present your data in the Chromatography Data Table. Explain why each tube was a certain color using the buffers used at each step.

Explain how a researcher could use the GFP protein in his/her research.

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