8.5: Part II- Separating the Green Fluorescent Protein with Column Chromatography
- Page ID
- 139112
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Reagents
- Microfuge tube rack with the microfuge tube of lysed cells (EC, from Part I of this lab)
- Containers with the following:
- Binding buffer (BB) = 4.0 M Ammonium sulfate solution
- Wash buffer (WB) = 1.3 M Ammonium sulfate solution
- Elution buffer (EB) = 10 mM Tris, 1 mM EDTA, pH 8.0 solution
- 20% Ethanol solution (For cleaning and storing resin in columns at the end of lab)
Equipment and Supplies
- 2 1.5-mL microfuge tubes
- Liquid waste container
- P-1,000 micropipette
- Tip box of disposable pipette tips
- Chromatography column
- Microcentrifuge (will be shared among all groups)
- Waste container (will be shared among groups)
Procedure
- Assign tasks in your group. Have one person make sure that your materials are ready (steps 2–3), another person set up the chromatography column (steps 4–5), and another person spin the lysed cells and remove the supernatant (step 6–7).
- Check to make sure that you have all the reagents listed.
- Label two clean microfuge tubes “SUPER” and “GFP.”
- Set up your chromatography column as directed by your instructor, being careful not to dislodge the stopcock attached to the lower portion of the tube. Do not allow the column to run dry.
- Prepare the column:
- Set the liquid waste collection container under the stopcock.
- Carefully open the column by turning the stopcock valve, and allow the liquid to drain into the waste collection container.
- Close the valve once there is about 1–2 mm of liquid left above the resin bed.
- Using the P-1000 micropipette and a new tip, add 1,000 μL (1 mL) of binding buffer (BB) gently down the side of the chromatography column.
- Open the column by turning the stopcock valve, and allow the liquid to drain into the waste collection container.
- Close the valve once there is about 1–2 mm of liquid left above the resin bed.
- Make sure that the liquid is no longer draining from the column into the waste container.
- Spin the EC tube in the microcentrifuge for five minutes to create a pellet of the cell debris.
- Examine the tube. You should see a supernatant and a solid pellet. Use the long wave UV light to examine your tube. What color is the supernatant? The pellet? What are the contents of each?
- Using the P-1,000 pipette, carefully remove 200 μL of EC supernatant without disturbing the cell debris pellet, and dispense the supernatant into the SUPER tube. If you dislodge the debris pellet, you will have to centrifuge the tube again.
- Using a new tip, add 200 μL of binding buffer (BB) to the SUPER tube. Mix by gently pumping the solution in and out.
- Using the same tip but changing the volume, add 400 μL of the SUPER tube mixture to the chromatography column. Carefully dispense the solution down the side of the column to avoid disturbing the surface of the resin bed.
- Open the valve and allow the solution in the column to drain into the waste collection container. Close the valve once there is about 1–2 mm of liquid left above the resin bed.
- Examine the column and locate the green fluorescent protein. Is it spread throughout the resin bed, or does it appear to be restricted to a single band? Record your observations in your notebook.
- Using a new tip, add 1,000 μL (1 mL) of wash buffer (WB) gently down the side of the chromatography column.
- Open the valve and allow the solution in the column to drain into the waste collection container. Close the valve once there is about 1–2 mm of liquid left above the resin bed.
- Examine the column with the long wave UV lamp and locate the green fluorescent protein. Has the location of the green fluorescent protein changed in the resin bed?
- Using a new tip, add 1,000 μL of elution buffer (EB) twice (2 mL total), gently, down the side of the chromatography column.
- Set the GFP tube under the stopcock. Open the valve and allow the part of the eluate that is fluorescing under UV light to drain into the GFP tube. Close the valve and cap the tube when done.
- Set the waste collection container back under the stopcock. Open the valve and allow the rest of the eluate to drain into the waste container. Close the valve once there is about 1–2 mm of liquid left above the resin bed.
- Using a new tip, add 1,000 μL of 20% ethanol three times (3 mL total) gently, down the side of the chromatography column.
- This time, open the valve and allow the solution in the column to drain into the waste collection container until there is about 1 cm of liquid left above the resin bed then close the valve securely.
- Now place the caps back on the column tightly to prepare it for storage.
- Pour the contents of the waste collection container down the sink drain.
Data Analysis
Compare your GFP tube with GFP tubes from other groups. Is there a difference in intensity of color from sample to sample under UV? Record your observations in your notebook.