Protein Fractionation and Purification

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Questions and Answers

Protein fractionation is essential for isolating a specific protein from a complex mixture. What is the MOST direct outcome of this process?

Obtaining a purified sample of the protein of interest for further analysis. (correct)
Increasing the overall entropy and disorder of the protein mixture.
Randomly denaturing most of the proteins to reduce sample complexity.
Determining the precise atomic structure of all proteins in the sample.

In protein fractionation, several properties can be exploited to achieve separation. If you wanted to separate proteins based solely on their size, which method would be MOST appropriate?

Affinity chromatography.
Ion exchange chromatography.
Isoelectric focusing.
Gel filtration chromatography. (correct)

When using salt precipitation for protein fractionation, increasing the salt concentration can lead to the precipitation of proteins. At what specific salt concentration does a given protein precipitate?

It is consistent across all proteins due to uniform structure.
It is universally high, because all proteins precipitate at same high salt concentrations.
It is unpredictable, because any salt concentration above zero can cause precipitation.
It depends uniquely on the protein's solubility characteristics. (correct)

In the context of protein precipitation using ammonium sulfate, what underlying principle explains why proteins become less soluble at high salt concentrations?

Salt ions compete with protein for water molecules, reducing protein solvation. (C) Signup and view all the answers

Dialysis is a protein purification technique. How does it work to separate proteins?

By using a semi-permeable membrane that allows small molecules to pass through, but retains larger proteins. (D) Signup and view all the answers

Protein electrophoresis separates proteins based on their charge-to-mass ratio. Which factor MOST directly affects the movement of a protein through the gel matrix?

Electric field strength. (B) Signup and view all the answers

In gel electrophoresis, what is the role of the gel matrix (e.g., polyacrylamide) in separating proteins?

It provides a frictional environment that separates proteins based on size and shape. (C) Signup and view all the answers

You are analyzing a gel after performing protein electrophoresis and notice that smaller proteins have migrated farther from the well compared to larger proteins. What is the MOST accurate interpretation of this observation?

Smaller proteins experience less resistance from the gel matrix. (D) Signup and view all the answers

During centrifugation, particles separate based on their physical properties. For a particle to sediment to the bottom of the tube, how must its density relate to the density of the surrounding solvent?

It must be higher than the solvent's density to overcome buoyancy. (B) Signup and view all the answers

Which type of centrifugation involves separating particles based on their sedimentation rate through a density gradient, leading to distinct bands?

Equilibrium density-gradient centrifugation. (A) Signup and view all the answers

How does chromatography achieve separation of molecules in a mixture?

By allowing molecules to distribute between a stationary and a mobile phase. (B) Signup and view all the answers

What determines the classification of chromatography as either liquid chromatography (LLC, LSC) or gas chromatography (GSC, GLC)?

The mobile phase used. (A) Signup and view all the answers

In thin-layer chromatography (TLC), what serves as the stationary phase?

A thin layer of adsorbent material on a flat surface. (A) Signup and view all the answers

Which type of chromatography separates molecules based on their specific biological affinity for a particular ligand?

Affinity chromatography. (A) Signup and view all the answers

Consider a scenario where a protein mixture is subjected to ion exchange chromatography. If the column is designed to bind negatively charged proteins, what type of resin is MOST likely used?

Anionic resin. (B) Signup and view all the answers

In gel filtration chromatography, molecular weight determination of macromolecules relies MOST directly on what property of the stationary phase?

Pore size. (C) Signup and view all the answers

Plasma protein fractionation is often used to isolate therapeutic proteins. If you want to obtain immunoglobulins from plasma, which initial step is MOST commonly used?

Centrifugation. (A) Signup and view all the answers

Which statement ACCURATELY describes the role of centrifugation in plasma protein fractionation?

It separates plasma into different layers based on density. (C) Signup and view all the answers

Cold ethanol fractionation is a method used in plasma protein purification. What principle underlies the use of cold ethanol in this process?

Ethanol reduces the dielectric constant of the solution, causing selective protein precipitation. (D) Signup and view all the answers

You are tasked with purifying a specific enzyme from a complex cellular extract. To begin, you need to disrupt the cells while maintaining the enzyme’s activity. Which method is MOST appropriate for this initial step?

Performing gentle cell lysis followed by homogenization. (B) Signup and view all the answers

After preparing a tissue extract, you need to determine the BEST approach for protein fractionation. Which factor should be considered to select the MOST appropriate technique to isolate the protein?

All of the above. (E) Signup and view all the answers

A researcher is using electrophoresis to separate proteins but observes broad, diffuse bands instead of sharp, well-defined bands. What adjustment is MOST likely to improve the resolution of the protein separation?

Use a gel with a higher percentage of crosslinking. (D) Signup and view all the answers

A protein sample is being prepared for gel filtration chromatography. What would be the MOST important consideration to ensure optimal separation?

The sample is diluted to reduce viscosity. (D) Signup and view all the answers

In affinity chromatography, after the target protein binds to the immobilized ligand, what is the MOST common method used to elute the bound protein?

Altering the pH or ionic strength. (B) Signup and view all the answers

A researcher aims to purify a protein with a known isoelectric point (pI) of 7.0 using isoelectric focusing. To ensure optimal focusing, what pH gradient range should the researcher establish?

A pH gradient centered around 7.0. (D) Signup and view all the answers

When performing salting out, a protein precipitates at a certain salt concentration. What is the effect of further increasing the salt concentration beyond this point?

The protein will remain precipitated, and other proteins may also precipitate. (B) Signup and view all the answers

A researcher is using dialysis to remove ammonium sulfate from a protein solution. Which specific property of the dialysis membrane is MOST crucial for the effective removal of the salt?

Pore size that allows the passage of salt but not the protein. (B) Signup and view all the answers

If a protein does not bind to the affinity column, what would happen?

The protein is washed away during the washing step. (B) Signup and view all the answers

In size exclusion chromatography, which molecules elute FIRST from the column?

The larger molecules. (D) Signup and view all the answers

Reverse phase chromatography is MOST useful for proteins with which of the following properties?

Hydrophobic regions on the surface. (D) Signup and view all the answers

Which type of chromatography is MOST effective for purifying a fusion protein tagged with a specific peptide sequence?

Affinity chromatography. (A) Signup and view all the answers

Why is it important to use mild conditions during cell lysis when preparing a protein extract for fractionation?

To minimize the modification of the desired protein. (B) Signup and view all the answers

Which of the following is a PRIMARY consideration when choosing a protein fractionation technique?

The abundance of the desired protein. (A) Signup and view all the answers

If you want to separate proteins based on structural characteristics, which technique would be appropriat?

Size exclusion chromatography. (D) Signup and view all the answers

What determines the separation of proteins during electrophoresis?

Mass and charge. (C) Signup and view all the answers

In a mixture of 3 proteins, if you perform differential centrifugation, what determines which protein precipitates first?

Size and density. (D) Signup and view all the answers

In immunoaffinity chromatography, which of the following is required?

Resin with a bound antibody. (B) Signup and view all the answers

Flashcards

Protein Fractionation

A process to isolate a single or multiple types of protein from a complex mixture.

Purpose of Protein Fractionation

Commercial products like enzymes, nutritional proteins, biopharmaceuticals, research, quantification, protein structure studies, and post-translational modifications.