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6: Activity 2-2 - Dialysis for Enzyme Purification

Last updated

Apr 19, 2025
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- 5: Activity 2-1 - Purification via Ammonium Sulfate Saturation of Cell-Free Extract
- 7: Activity 2-3 - Size Exclusion Gel Filtration Purification of Enzymes

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Learning Objectives

Explain the purpose of dialysis in enzyme purification.
Describe how a semipermeable membrane works in dialysis.
Identify what components are retained vs. removed during dialysis.
Understand how ammonium sulfate affects protein solubility and why its removal is necessary.
Predict how impurities like salts may interfere with downstream assays.

Definition: Term

Dialysis: A method used to remove small molecules (e.g., salts) from a solution containing larger macromolecules (e.g., proteins), using a semi-permeable membrane.
Semi-permeable Membrane: A material with pores small enough to allow passage of small molecules while retaining larger ones.
Ammonium Sulfate Saturation: A method to precipitate proteins based on their solubility in high salt concentrations.
Cell-Free Extract (CFE): A lysate from cells where all cellular structures are broken down, leaving behind soluble proteins and other molecules.
Extraction Buffer: A solution that maintains protein stability during purification by controlling pH and ionic strength.
Bradford Assay: A colorimetric assay used to measure total protein concentration using Coomassie Brilliant Blue dye.
Beer’s Law (A = εcl): A formula that relates absorbance to concentration, path length, and molar extinction coefficient—used to quantify specific proteins like P450.

Guiding Questions:

Why might we want to remove ammonium sulfate before analyzing the enzyme?
How does the dialysis membrane know which molecules to let through?
What would happen if you didn't stir the buffer during dialysis?
What might a failed dialysis look like during a downstream assay?
Why do we freeze the samples after dialysis instead of using them immediately?

Dialysis for Enzyme Purification

In this lab, we are continuing the purification of the P450 enzyme from a cell-free extract using a technique called dialysis. At this stage, the enzyme has already been partially purified using ammonium sulfate saturation, which helps precipitate out proteins based on their solubility. However, the remaining solution still contains excess ammonium sulfate and other small molecules that we need to remove before we can further analyze the enzyme.

Dialysis works like a molecular sieve or a "tea bag for molecules." Imagine placing a mixture of small and large molecules into a special bag made of a semi-permeable membrane—this membrane has tiny pores that allow small molecules like salts and buffer components to pass through while trapping larger molecules like proteins inside. When you submerge the bag in a clean buffer solution, the small molecules diffuse out, but the proteins stay in. By repeatedly changing the surrounding buffer, we gradually remove unwanted small molecules while keeping our enzyme safe and contained.

This method is essential because the presence of salts like ammonium sulfate can interfere with downstream applications such as protein concentration measurements or enzyme activity assays. After dialysis, your enzyme samples will be in a clean extraction buffer and ready for quantification and functional testing.

Materials:

Pipettes and tips
Frozen protein fractions (from previous lab)
Dialysis tubing (soaked in ddH₂O in a 100 mL beaker)
Dialysis clips (Carolina #684216 and #684239)
2 L beaker containing 1 L cold Extraction Buffer with a magnetic stirrer
COLD Extraction Buffer: 50 mM potassium phosphate, pH 7.4 OR 0.25X PBS
- Prepared from from 1 M stock:
  - 95 g of monobasic potassium phosphate (Carolina #884250)
  - 52.5 g of dibasic potassium phosphate (Carolina #884300)

Procedure:

Thaw frozen fractions from the previous lab completely on ice.
Take and prepare a piece of dialysis tubing from the water-filled beaker.
1. Tie one end securely with a dialysis clip or a tight knot.
2. Carefully pipette your protein fraction into the open end of the tubing.
3. Remove any air bubbles by gently squeezing the tubing.
4. Tie off the open end and secure it with a second dialysis clip.
5. Label the clip or tubing clearly for identification.
Place the sealed dialysis tubing with your samples in a 2 L beaker containing 1 L of cold Extraction Buffer on a magnetic stirrer.
Let it stir gently for 1 hour.
After 1 hour, replace the buffer with fresh Extraction Buffer and stir for another hour.
After dialysis, transfer the contents of each tubing into a clean, labeled 1.5 mL centrifuge tube. Label as "Dialysis, initial, date"
Transfer a 50 µL aliquot from each fraction, label as "Dialysis-SDS, initial date"
Store the samples in a freezer until further use.

Learning Objectives

After completing the lab and associated analysis, students should be able to:

Perform dialysis to purify an enzyme sample.
Explain the outcome of dialysis based on visual and analytical results.
Accurately measure protein concentration using the Bradford assay.
Use Beer’s Law to determine P450 concentration from absorbance data.
Evaluate enzyme purity and functionality through specific activity calculation.
Interpret purification data and conclude each fraction's quality.

Key Takeaways:

Dialysis is a simple yet powerful technique that removes contaminants without harming the protein.
Buffer composition and temperature matter in preserving enzyme structure and activity.
Proper labeling and sample handling are essential to track purification progress.
Quantitative techniques like the Bradford assay and Beer’s Law provide insight into the effectiveness of purification.
Comparing fractions allows identification of the most enriched and active enzyme preparation.

Post-Lab Questions:

Which fraction had the highest specific activity, and why?
What evidence suggests that dialysis was successful?
Did the protein concentration decrease or increase after dialysis? What does that tell you?
If the P450 concentration didn't change, but activity decreased, what might that imply?
How might you improve dialysis efficiency if you were to repeat this experiment?

Note

Next Lab Tasks: We will use the the dialyzed fractions to perform the following analyses:

Measure total protein concentration using the Bradford Assay.
Quantify P450 concentration using Beer’s Law (A = εcl).
Determine enzyme activity (Rate of reaction in µM/sec/mg protein).

Learning Objectives

Definition: Term

Guiding Questions:

Dialysis for Enzyme Purification

Materials:

Procedure:

Learning Objectives

Key Takeaways:

Post-Lab Questions:

Note

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