Question

You are purifying a protein for the first time. You have solubilized it with homogenization in a blender followed by differential centrifugation. You wish to try ammonium sulfate precipitation as the next step. Knowing nothing beforehand about the amount of ammonium sulfate to add, design an experiment to find the proper concentration (\% saturation) of ammonium sulfate to use.

Short answer

Prepare a series of ammonium sulfate solutions with different concentrations, mix with protein solution, incubate, centrifuge, measure protein concentration, and identify optimal precipitation concentration.

Step-by-step solution

Prepare Protein Solution

Start by preparing the homogenized protein solution using a blender and following up with differential centrifugation to remove cell debris and other larger particles.

Create Gradient Solutions

Prepare a series of ammonium sulfate solutions with different saturation percentages. For example, you could use 20%, 40%, 60%, 80%, and 100% saturation solutions.

Add Ammonium Sulfate

Aliquot equal volumes of the protein solution into separate test tubes and add the different concentrations of the ammonium sulfate solutions to each test tube.

Incubate and Centrifuge

Incubate each test tube at room temperature for a sufficient amount of time (typically 30 minutes to 1 hour) to allow protein precipitation. Then centrifuge the tubes to pellet the precipitated proteins.

Measure Protein Concentration

Carefully remove the supernatant from each test tube and determine the amount of protein in each pellet. This can be done using a protein quantification assay like Bradford or BCA assay.

Analyze Data

Plot the protein concentration as a function of ammonium sulfate concentration. Identify the concentration at which the maximum amount of protein is precipitated without significant loss of protein in the supernatant.

Key concepts

Ammonium Sulfate Precipitation

Ammonium sulfate precipitation is a method used to purify proteins by exploiting their solubility properties. When you add ammonium sulfate, it changes the solubility of proteins, causing them to precipitate out of the solution.

Here’s how it works:

• Ammonium sulfate disrupts the hydrogen bonds and hydrophobic interactions in proteins.
• This causes proteins to become less soluble and precipitate out of the solution as the salt concentration increases.

You prepare a series of solutions with different concentrations of ammonium sulfate. When the protein solution is mixed with these, proteins will precipitate at different salt concentrations.

This method helps in concentrating the protein and removing other contaminants. By experimenting with varying concentrations, you can determine the optimal saturation that precipitates the target protein most effectively.

Differential Centrifugation

Differential centrifugation is a technique used to separate components in a mixture based on size and density. This method helps in the initial purification steps of your protein.

Here's how it’s typically done:

• You start by homogenizing the tissue sample using a blender to break down the cells.
• The homogenate is then spun at low speed to remove heavier cell debris and organelles.

The supernatant, which contains smaller particles like proteins, is collected and spun at higher speeds to separate out different cellular components.

This allows you to obtain a cleaner solution containing your protein of interest, which can then be further purified using other methods like ammonium sulfate precipitation.

Protein Quantification

Protein quantification is essential to determine how much protein you have after each purification step. Knowing the concentration helps in analyzing the effectiveness of your purification process.

Common methods of protein quantification include:

• The Bradford Assay: Uses Coomassie Brilliant Blue dye, which binds to proteins and causes a color change that can be measured spectrophotometrically.
• The BCA Assay: Relies on the reduction of Cu^2+ to Cu^1+ by protein in an alkaline medium, leading to a color change measured spectrophotometrically.

You take a small sample from each test tube and run these assays to get the protein concentration in each pellet.

By plotting the data, you can figure out at which ammonium sulfate concentration your protein precipitated most effectively, aiding in the optimization of the purification process.