SDS - PAGE

Summary

In this video lecture, the concept of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is explained as a technique for determining the molecular weights of proteins. Unlike native PAGE, which preserves proteins in their natural state, SDS-PAGE denatures proteins using the detergent SDS, causing them to unfold and separate into subunits. This process eliminates the influence of protein charge, allowing proteins to be separated based solely on their size. The video also discusses the addition of reducing agents to break disulfide bonds and highlights the practical applications of both native PAGE and SDS-PAGE.

Takeaways

• 😀 Native PAGE preserves the original structure of proteins and allows the study of their biological activity.
• 😀 SDS-PAGE is used to determine the molecular weights of proteins and to analyze the subunit composition of proteins.
• 😀 SDS stands for sodium dodecyl sulfate, an anionic detergent that imparts a negative charge to proteins.
• 😀 Proteins treated with SDS undergo denaturation, losing their native structure and unfolding into rod-like shapes.
• 😀 SDS molecules coat proteins, masking their original charges and making them uniform in charge-to-mass ratio.
• 😀 A reducing agent like beta-mercaptoethanol is used to break disulfide bridges in proteins, aiding their denaturation.
• 😀 SDS-PAGE separates proteins based on their size or molecular weight, with larger proteins migrating slower.
• 😀 Smaller proteins in SDS-PAGE migrate faster through the gel due to their lower molecular weight.
• 😀 Native PAGE separates proteins based on their charge density, size, and shape, rather than molecular weight.
• 😀 SDS-PAGE is primarily used for determining molecular weights, while native PAGE is used for detecting enzymes.
• 😀 The video emphasizes the distinction between native PAGE and SDS-PAGE in terms of the types of protein analysis they are best suited for.

Q & A

What is native PAGE and how does it work?

Native PAGE (Polyacrylamide Gel Electrophoresis) is a method used to separate proteins based on their charge, size, and shape without denaturing them. In this technique, proteins remain in their native form, allowing for the study of their biological activity.

What is the main purpose of SDS-PAGE?

SDS-PAGE is used to determine the molecular weight of proteins in a sample and to assess whether a protein is composed of single or multiple subunits. This technique involves denaturing proteins with SDS and then separating them based on size or molecular weight.

What does SDS stand for, and how does it affect proteins?

SDS stands for Sodium Dodecyl Sulfate, an anionic detergent. It disrupts the native structure of proteins by denaturing them, causing the protein to unfold and its subunits to separate. SDS also coats the proteins with a negative charge, masking the proteins' original charges.

How does SDS interact with proteins on a molecular level?

SDS molecules associate with proteins by attaching their hydrophobic tail to every two amino acid residues. This coating masks the protein's original charges and results in a uniform charge-to-mass ratio for all proteins, which is essential for their separation during electrophoresis.

Why is a reducing agent like beta-mercaptoethanol added in SDS-PAGE?

Beta-mercaptoethanol is added to SDS-PAGE to break any disulfide bonds that may be present in the native structure of proteins. This helps to fully denature the protein and ensures that all subunits are separated, allowing for accurate size determination.

How does the size of a protein affect its migration during SDS-PAGE?

In SDS-PAGE, proteins are separated based on their size or molecular weight. Larger proteins have a higher molecular weight and move slower through the gel, while smaller proteins migrate faster.

What is the key difference between native PAGE and SDS-PAGE?

Native PAGE preserves the protein’s native form and separation is based on a combination of charge, size, and shape. In contrast, SDS-PAGE denatures proteins using SDS and separation is based solely on molecular weight.

What kind of samples is native PAGE typically used for?

Native PAGE is commonly used for separating and detecting enzymes, as it preserves the proteins' native conformation and biological activity.

What does SDS-PAGE tell us about a protein's structure?

SDS-PAGE provides information about the molecular weight of proteins, and can also reveal whether a protein is a single subunit or composed of multiple subunits, based on how the protein migrates through the gel.

Why is SDS-PAGE the most popular form of protein electrophoresis?

SDS-PAGE is widely used because it effectively denatures proteins, leading to consistent separation based on size, and it can be used to determine molecular weights of proteins, making it essential for protein characterization.