AH Biology - Unit 1 - KA1(c) - Separation Techniques

What is a centrifuge?

A centrifuge is a piece of equipment that spins a suspension or solution at a high speed. They separate substances in a solution or suspension by their density.

When using a centrifuge, what do the less dense substances do?

When using a centrifuge, the less dense substances remain in the supernatant at the top.

When using a centrifuge, what do the more dense substances do?

When using a centrifuge, the more dense substances settle at the bottom to form the pellet.

What is paper and thin layer chromatography?

Paper and thin chromatography is a type of chromatography:

• Used to separate mixtures of sugars and amino acids.

• Uses solubility to separate them.

• A solvent is run through paper or a gel (TLC)

• A chromatogram is produced.

• The further a solute travels along the chromatogram, the more soluble the substance is in the substance used.

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What is affinity chromatography?

Affinity chromatography is a type of chromatography used to isolate specific target proteins from a liquid mixture. It relies on using substances that have specific relationships with each other. For example:

• Enzyme and substrate

• Antigen and antibody

• Receptor and ligand

What are the stages of affinity chromatography?

The phases of affinity chromatography are:

1. A solid matrix or gel column is created with a specific molecule bound to the gel.

2. The mixture containing the target protein is passed down the column.

3. The soluble, specific target protein binds with their specific molecule (the molecule attached to the gel in stage 1), because they have a high affinity for them.

4. Other non-target molecules are washed out because they have a weaker affinity for the molecule bound to the gel.

What is the first stage of affinity chromatography?

The first stage of affinity chromatography is “A solid matrix or gel column is created with a specific molecule bound to the gel.”

What is the second stage of affinity chromatography?

The second stage of affinity chromatography is “The mixture containing the target protein is passed down the column.”

What is the third stage of affinity chromatography?

The third stage of affinity chromatography is “The soluble, specific target protein binds with their specific molecule (the molecule attached to the gel in stage 1), because they have a high affinity for them.”

What is the fourth stage of affinity chromatography?

The fourth stage of affinity chromatography is “Other non-target molecules are washed out because they have a weaker affinity for the molecule bound to the gel.”

What does gel electrophoresis do and how does it work?

• Gel electrophoresis separates proteins or nucleic acid by running a current to produce an electric field across a gel matrix.

• The wells at the bottom edge of the gel plate are filled with the mixture containing the target molecules.

• When the electric field is created, the charged macromolecules move through the field at different rates.

What are the two main types of gel electrophoresis?

The two main types of gel electrophoresis are:

• Native gels

• SDS-PAGE

What do native gels separate proteins by, and why?

Native gels separate proteins by their size, shape, and charge because they do not denature the molecule.

What do SDS-PAGES separate proteins by, and why?

SDS-PAGES separate proteins by size alone because they denature all the molecules and give them an equal charge (negative).

In gel electrophoresis, what do larger molecules do in comparison with smaller molecules?

In gel electrophoresis, larger molecules move slower than smaller molecules.

In gel electrophoresis, what do charged molecules do?

In gel electrophoresis, charged molecules move towards the opposite charge.

What is isoelectric point (IEP) used to separate?

Isoelectric point (IEP) is used to separate proteins from a liquid mixture.

What is the isoelectric point?

The isoelectric point is the pH at which a soluble protein:

• Has no net charge.

• Precipitates out of solution.

Since different proteins have different isoelectric points, what does adding a specific buffer with a particular pH ensure?

Since different proteins have different isoelectric points, adding a specific buffer with a particular pH ensures that only certain proteins will precipitate out of solution.

What is the process of separating mixtures using isoelectric point and electrophoresis?

During the process of separating mixtures using isoelectric point and electrophoresis:

• An electric current and a pH gradient are applied across the gel.

• This stops the protein from migrating through the gel when it reaches its isoelectric point in the pH gradient. This is because at this point, the protein has no net charge.

When would a protein with an isoelectric point of four precipitate out of solution?

A protein with an isoelectric point of four would precipitate at pH four. add pic