7: Activity 2-3 - Size Exclusion Gel Filtration Purification of Enzymes
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- Define gel filtration chromatography and explain how it separates molecules by size.
- Describe the physical structure and function of Sephadex G-25 beads.
- Predict elution order based on protein size in the context of an exclusion limit.
- Understand the purpose of this technique in the multi-step purification of Cytochrome P450BM3.
- Identify materials and prepare the gel filtration column correctly.
- Chromatography: A technique for separating mixtures based on physical or chemical properties like size, charge, or affinity.
- Gel Filtration Chromatography: A type of chromatography that separates molecules based on size using porous beads; also called size-exclusion chromatography.
- Exclusion Limit: The maximum molecular weight that can enter the pores of a gel bead; molecules larger than this size are excluded and elute faster.
- Sephadex G-25: A type of gel filtration matrix made from dextran beads with pores that exclude proteins larger than ~25 kDa.
- Elution: The process by which molecules exit the chromatography column and are collected in fractions.
- Cytochrome P450BM3: A heme-containing bacterial enzyme, often red-brown in color, used here as a model for protein purification studies.
- Molecular weight (kDa) and how it affects protein diffusion.
- Previous lab techniques: ammonium sulfate precipitation, dialysis, Bradford assay, and Beer’s Law.
- Why it’s important to purify proteins in biotechnology and research settings.
Gel Filtration Chromatography
Chromatography is a powerful technique used to separate complex mixtures of biological molecules based on their physical and chemical properties. In this activity, we will focus on gel filtration chromatography—a type of chromatography that separates proteins based on their size. This method is also called size-exclusion chromatography. Previously, you began purifying the enzyme Cytochrome P450BM3 from E. coli by using ammonium sulfate precipitation, which allowed you to separate some soluble proteins based on solubility. You then dialyzed those protein fractions to remove small molecules like salts. Now, we’ll use gel filtration to take the purification process a step further.
Gel filtration uses a column packed with tiny porous beads made of a polymer like Sephadex. Think of these beads as sponges with tunnels inside them. When a mixture of proteins is poured onto the column, the proteins take one of two general routes:
- Large proteins, like Cytochrome P450BM3, are too big to fit into the pores of the beads. Imagine a beach ball rolling over a sponge—it doesn’t sink in, so it moves around the beads and flows through the column faster.
- Small proteins, on the other hand, enter the pores—like marbles sinking into the sponges—so they take a longer, winding path through the beads and come out later. This is why larger molecules elute (come out) first, and smaller ones elute later.
Common misconception: In gel filtration chromatography, molecules are separated by size, but not in the way students often assume. The Sephadex G-25 column used here has an exclusion limit of 25 kDa. Molecules smaller than 25 kDa can enter the pores in the gel beads, so they take a longer, winding path through the column and elute later. Molecules larger than 25 kDa are excluded from the pores and take a more direct route around the beads, eluting earlier. Now, here’s where it gets interesting: while both 60 kDa and 120 kDa proteins are too large to enter the beads, the 120 kDa protein elutes slightly faster than the 60 kDa one. This is because larger molecules interact even less with the gel and diffuse through the column more directly. So yes, both are excluded, but the largest molecules still come out first. So, in gel filtration, the more excluded a molecule is, the faster it elutes. The biggest ones race out first—not because they’re fast, but because they’re too big to get delayed.
Materials Needed
- Make sure you have all materials and protocol from your previous labs on:
- Bradford Assay
- Beer’s Law
- Enzyme Kinetics
- Gel filtration column with stopcock
- Sephadex-G25 beads (Sigma-Aldrich, #G25150-50G)
- Dialyzed reddish sample from the previous lab
- Equilibration buffer: 100 mM potassium phosphate, pH 7.4 OR 0.5X PBS
- Prepared from 1 M stock solution:
- 95 g monobasic potassium phosphate (Carolina, #884250)
- 52.5 g dibasic potassium phosphate (Carolina, #884300)
- Prepared from 1 M stock solution:
- 1.5 mL microcentrifuge tubes
- 15 mL conical centrifuge tube
- Waste container (for collecting flow-through from the column)
- Ice bucket for keeping fractions cold
Procedure
Prepare Your Gel Filtration Column
- Weigh out 1 g of Sephadex-G25 beads.
- Add the beads to 10 mL of 100 mM potassium phosphate buffer in a 15 mL conical tube.
- Gently invert the tube continuously for 5 minutes to fully hydrate and mix the beads.
- Carefully pour the bead suspension into the gel filtration column until it's about half-full (~50%).
- Let the buffer drain slowly through the column, allowing the beads to settle and pack. Once settled, close the stopcock.
Run Your Sample
- Take your reddish dialyzed protein sample from the freezer and allow it to thaw on ice.
- Gently pipette the entire thawed reddish sample onto the top of the column matrix.
- Wait for the meniscus to reach the top of the settled bead bed, then close the stopcock.
- Add 300 µL of buffer at a time to the top of the column.
- For each addition, collect the liquid that comes out into a new 1.5 mL microcentrifuge tube.
- Place each collected tube on ice to keep the proteins stable.
- Repeat this step (2-6) until the liquid coming out is colorless.
Combine the Purified Fractions
- Line up your collection tubes against a sheet of white paper to see which fractions look reddish.
- These reddish fractions likely contain Cytochrome P450BM3, which has a natural red-brown heme color.
- Combine the reddest fractions into a single microcentrifuge tube.
- Measure and record the total volume of this combined fraction. Congratulations! You’ve successfully completed gel filtration purification of Cytochrome P450BM3.
- Take a 50 µL aliquot of your purified fraction, transfer it into a new labeled microcentrifuge tube, and freeze it.
- Label the tube as: "Pure WB - initial, [Your Name], [Date]"
8. Analyze Your Purified Enzyme
- Using the remaining volume of your purified sample:
- Determine total protein concentration using the Bradford Assay.
- Measure Cytochrome P450BM3 concentration using Beer’s Law (based on absorbance at the Soret peak).
- Measure the enzymatic activity of P450BM3 using your kinetic assay method.
- Create a data table. Record your observations, volumes, concentrations, and calculations
Data Table:
| Fraction | Protein conc. (µg/µL) | P450 conc. (µM) | Rate (µM/s/mg) |
|---|---|---|---|
| CFE A | |||
| CFE B | |||
| Supernatant | |||
| 0–40% Pellet | |||
| 40–60% Pellet | |||
| 40–60% Pure |
After completing the activity, you should now be able to:
- Describe how gel filtration separates proteins by size and interpret real elution data.
- Identify which fractions contain your protein of interest based on color and elution order.
- Explain why larger proteins elute faster in gel filtration, despite intuition suggesting smaller should move faster.
- Analyze and report protein concentration, purity, and enzymatic activity after purification.
- Integrate gel filtration as one step in a broader multi-technique protein purification strategy.
- Why do large proteins like P450BM3 elute before smaller ones in gel filtration chromatography?
- What would happen if we used a Sephadex with a much higher exclusion limit (e.g., G-100)?
- How does this purification step complement the ammonium sulfate precipitation you did previously?
- In your elution data, did you observe fractions that clearly contained the reddish P450 protein? What does this suggest about its size?
- Did the protein concentration correlate with the reddish color? Why or why not?
Suggested Summary Table to Create (Example)
| Fraction ID | Color | Protein Concentration (µg/µL) | P450 Conc. (µM) | Enzyme Rate (µM/s/mg) | Notes |
|---|---|---|---|---|---|
| 40–60% Pure | Reddish | 0.95 | 1.2 | 0.03 | Final purified sample |
| Supernatant | Faint | 0.40 | 0.1 | - | Likely salt/impurities |
