Eletroforese e Cromatografia de Proteínas

Summary

In this educational video, the speaker explains two techniques used to separate proteins: electrophoresis and chromatography. Electrophoresis, a method developed in 1937, uses electricity to separate proteins based on their charge and molecular mass. The speaker demonstrates the process using venom from the jararaca snake, emphasizing the importance of protein research for developing antivenoms. Additionally, the video covers chromatography, a technique pioneered in 1903, which separates proteins by their affinity for different phases. The speaker highlights the significance of these techniques in scientific research, particularly in toxin and antivenom development.

Takeaways

• 😀 Proteins are essential macromolecules vital for all living organisms, and the venom of the Jararaca snake is composed of 90% proteins.
• 😀 The process of protein separation can be achieved through various techniques, including protein electrophoresis and chromatography.
• 😀 Electrophoresis involves the migration of particles with charge (either positive or negative) in a liquid medium under the influence of an electric field.
• 😀 In the electrophoresis experiment, proteins from the Jararaca snake venom are separated based on their molecular mass after being treated with SDS, which gives all proteins a negative charge.
• 😀 A molecular weight marker or protein standard is used in electrophoresis to estimate the molecular mass of unknown proteins by comparison.
• 😀 The gel used in electrophoresis is made of polyacrylamide and acts as a molecular sieve, allowing smaller proteins to migrate faster than larger ones.
• 😀 Proteins are separated by their molecular mass during electrophoresis, with smaller proteins moving faster through the gel.
• 😀 The experiment requires a power source to generate an electric current, causing the proteins to migrate through the gel toward the positive pole.
• 😀 After the electrophoresis run, the gel is stained with a dye (like Coomassie Blue) to visualize the proteins, which are then analyzed based on their migration patterns.
• 😀 Chromatography, another method for protein separation, involves using a stationary phase (such as a resin) and a mobile phase (like an aqueous buffer) to separate proteins based on their affinity for the stationary phase.
• 😀 In chromatography, proteins with greater affinity for the stationary phase will stay longer, while those with lesser affinity will elute (move out) first, aiding in protein separation.

Q & A

What are proteins and why are they important?

Proteins are macromolecules essential for all living organisms. For example, the venom of the jararaca snake is composed of about 90% proteins, highlighting their critical role in functions such as defense and prey management.

What is the primary method to separate proteins?

Proteins can be separated using methods like protein electrophoresis and chromatography. These techniques help purify and analyze proteins from mixtures, such as snake venom.

What is electrophoresis and how does it work?

Electrophoresis refers to the migration of charged particles, like proteins, through a liquid medium under the influence of an electric field. Proteins are separated based on their charge and molecular mass.

What is the historical significance of electrophoresis?

Electrophoresis was first developed in 1937 by Swedish biochemist Arne Sörens, marking the beginning of a technique that is now widely used for protein separation and analysis.

Why is SDS added to the protein mixture during electrophoresis?

Sodium dodecyl sulfate (SDS) is added to ensure that all proteins acquire a uniform negative charge, allowing them to be separated by their molecular mass rather than their individual charges.

What is the purpose of the gel in protein electrophoresis?

The gel serves as a molecular sieve, separating proteins based on their size. Smaller proteins migrate faster through the gel, while larger proteins move more slowly.

What is the role of the molecular weight standard in electrophoresis?

A molecular weight standard is used to estimate the size of unknown proteins by comparing their migration patterns to those of proteins with known molecular weights.

What happens after the electrophoresis process is complete?

After the electrophoresis run, the gel is stained with a dye like Coomassie Blue to visualize the separated protein bands. The gel is then decolorized to remove excess stain for clearer results.

What is chromatography and how does it differ from electrophoresis?

Chromatography is a technique used to separate mixtures, such as proteins, based on their interactions with two phases: a stationary phase and a mobile phase. In contrast to electrophoresis, it focuses on interactions with the stationary phase rather than electrical charge or molecular mass.

What are the main differences between manual and automated chromatography?

Manual chromatography involves manually applying samples to a column, whereas automated chromatography, like high-efficiency liquid chromatography (HPLC), uses machines to streamline and optimize the process of separating proteins.