Video Animasi dan Aktivitas Lobaratorium: SDS PAGE

Summary

This video provides a comprehensive guide to SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), a powerful technique for protein analysis. It explains how proteins are denatured using SDS and beta-mercaptoethanol, separated by size in a polyacrylamide gel, and visualized using stains like Coomassie Brilliant Blue. The video also covers sample preparation, gel formation, electrophoresis setup, and troubleshooting tips, offering an in-depth look at the process. Additionally, it discusses how protein migration is influenced by molecular weight and how molecular markers help determine protein sizes. This technique is essential for protein research and purification.

Takeaways

• 😀 SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a powerful technique used to separate proteins based on their molecular weight.
• 😀 Proteins are denatured using SDS, which disrupts hydrogen bonds, hydrophobic interactions, and ionic bonds, and a reducing agent like beta-mercaptoethanol cleaves disulfide bonds.
• 😀 The protein samples are mixed with a loading buffer containing SDS, glycerol, bromophenol blue, and beta-mercaptoethanol, and heated at 95°C for 5 minutes.
• 😀 SDS binds uniformly to proteins, imparting them with a net negative charge, which allows them to be separated in the polyacrylamide gel.
• 😀 The gel is composed of two parts: a separating gel (pH 8.8) and a stacking gel (pH 6.8), which are made using acrylamide, bisacrylamide, ammonium persulfate, and TEMED.
• 😀 The polymerization of the gel involves free radical reactions, initiated by ammonium persulfate and accelerated by TEMED, which links acrylamide monomers into a cross-linked polymer network.
• 😀 Proteins are loaded into the gel and separated based on size as an electric field is applied, with larger proteins migrating more slowly than smaller ones.
• 😀 The stacking gel focuses proteins into a narrow band before they enter the separating gel, where proteins are separated based on molecular weight.
• 😀 During electrophoresis, glycine behaves as a neutral molecule in the stacking gel, while chloride ions move faster, concentrating the proteins in a defined area.
• 😀 After electrophoresis, the gel can be stained using Coomassie Blue to visualize protein bands, with molecular weight markers helping to estimate the size of unknown proteins.

Q & A

What is the primary purpose of SDS-PAGE in protein analysis?

The primary purpose of SDS-PAGE is to separate proteins based on their molecular weight, allowing researchers to analyze protein composition, estimate the size of proteins, and verify protein purity.

What role does SDS play in SDS-PAGE?

Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by disrupting hydrogen bonds, hydrophobic interactions, and ionic bonds, leading to linearized proteins with a uniform negative charge.

Why is β-mercaptoethanol added to the protein samples in SDS-PAGE?

β-mercaptoethanol is used as a reducing agent to cleave disulfide bonds in proteins, which helps in fully denaturing the proteins and ensuring they are linearized for proper separation during electrophoresis.

What is the function of the stacking gel in SDS-PAGE?

The stacking gel ensures that proteins enter the separating gel at the same time, preventing smeared bands by concentrating proteins into a narrow zone before they migrate into the separating gel, where they are separated by size.

How does the molecular weight of proteins affect their migration in SDS-PAGE?

Smaller proteins migrate faster through the polyacrylamide gel because they experience less resistance, while larger proteins are slower due to their size, allowing them to be separated based on their molecular weight.

What is the significance of the running buffer in SDS-PAGE?

The running buffer, typically Tris-glycine, helps conduct the electric current through the gel and maintains the pH required for optimal protein migration, ensuring proper protein separation and preventing buffer-related issues.

Why is bromophenol blue included in the sample loading buffer?

Bromophenol blue is a dye used to visualize the sample in the well. It also helps track the progress of the electrophoresis run by migrating ahead of the protein bands.

What is the purpose of using a molecular weight size marker in SDS-PAGE?

A molecular weight size marker consists of proteins of known sizes and is used to estimate the molecular weight of proteins in the samples by comparing the migration distance of the sample proteins to that of the marker.

What happens if the gel run in SDS-PAGE is stopped too early?

If the gel run is stopped too early, the protein bands may not be fully resolved, making it difficult to obtain clear results and accurately determine the protein sizes.

Why are proteins stained after SDS-PAGE, and how is it done?

Proteins are stained after electrophoresis to visualize the protein bands. Commonly, Coomassie Brilliant Blue is used, as it binds non-specifically to proteins and produces blue bands on a clear background, allowing for protein identification and analysis.