Question

How does the gel for isoelectric focusing differ from the gel for traditional electrophoresis? A. Isoelectric focusing uses a gel with much larger pore sizes to allow for complete migration. B. Isoelectric focusing uses a gel with SDS added to encourage a uniform negative charge. C. Isoelectric focusing uses a gel with a pH gradient that encourages a variable charge. D. The gel is unchanged in isoelectric focusing; the protein mixture is treated before loading.

Short answer

C: Isoelectric focusing uses a gel with a pH gradient.

Step-by-step solution

Understand the Concepts

Know the difference between isoelectric focusing and traditional electrophoresis. Isoelectric focusing separates proteins based on their isoelectric point (pI), while traditional electrophoresis separates them based on size and charge.

Analyze Each Option

Examine each answer choice to determine the correct distinction between the gels used.

Evaluate Option A

Option A suggests isoelectric focusing uses a gel with much larger pore sizes. This doesn't relate to the principle of isoelectric focusing, which focuses on pH gradients.

Evaluate Option B

Option B suggests isoelectric focusing uses a gel with SDS. Isoelectric focusing doesn't use SDS since it focuses on separating proteins based on their pI values, not their charge.

Evaluate Option C

Option C suggests that isoelectric focusing uses a gel with a pH gradient. This is correct because isoelectric focusing relies on a pH gradient to separate proteins based on their isoelectric points.

Evaluate Option D

Option D implies that the gel is the same for both methods, which is incorrect; isoelectric focusing gels have a pH gradient, unlike traditional electrophoresis gels.

Select the Correct Answer

Based on evaluations, the correct answer is C. Isoelectric focusing uses a gel with a pH gradient that encourages a variable charge.

Key concepts

Electrophoresis

Electrophoresis is a laboratory technique used to separate charged molecules, such as proteins or DNA, based on their size and electrical charge. This process involves placing the sample on a gel and applying an electric current. Due to the electric field, the molecules will migrate through the gel at different rates depending on their individual properties. Traditional electrophoresis often uses a gel like agarose or polyacrylamide and can be used to visualize DNA, RNA, or protein fragments. The technique can be essential in various fields such as molecular biology, biochemistry, and genetics.

• Charge: Molecules with different charges move towards the positive or negative electrode.
• Size: Smaller molecules move faster through the gel pores.
• Medium: Typically uses a gel that acts as a sieve to separate molecules.

By understanding electrophoresis, you can better appreciate the nuances of advanced methods like isoelectric focusing.

pH Gradient

A pH gradient is crucial to the process of isoelectric focusing. This gradient is established within the gel using a mixture of acids and bases, creating a range of pH values from one end of the gel to the other. The purpose of this gradient is to separate molecules based on their isoelectric point (pI), which is the pH at which a particular molecule carries no net electric charge. When an electric field is applied, molecules will migrate to the region in the gel where the pH matches their pI.

• Creating the Gradient: The gel is infused with a mix of acids and bases to produce a continuous pH range.
• Molecular Behavior: Proteins or other charged molecules move until they reach zero net charge.
• Stationary Phase: This is where the molecules will stop migrating, concentrating in specific areas.

Understanding the pH gradient helps to clarify why isoelectric focusing can effectively separate proteins with high precision.

Isoelectric Point

The isoelectric point (pI) is a fundamental concept in biochemistry, defining the pH at which a molecule carries no net electric charge. Different proteins have unique pI values based on their amino acid composition. Isoelectric focusing exploits this property to separate proteins within a sample.

• Isoelectric Point (pI): The specific pH where a molecule has zero net charge.
• Protein Separation: In isoelectric focusing, proteins migrate through the pH gradient until they reach a pH that matches their pI.
• Unique Profiles: Each protein concentrates at its pI, enabling detailed analysis.

Grasping the concept of isoelectric point makes it clearer how isoelectric focusing separates proteins based on their specific pI values, rather than their size or overall charge.