SDS PAGE : How does it works?

Animated biology With arpan
16 Jun 201906:16

Summary

TLDRIn this video, the host explains SDS-PAGE (SDS polyacrylamide gel electrophoresis) in under five minutes. SDS-PAGE is a technique used to separate proteins based on their molecular weight. The video covers the difference between stacking and resolving gels, the role of SDS and beta-mercaptoethanol in denaturing proteins, and how proteins are separated using electrophoresis. The process involves various components like chloride and glycine ions. Finally, the host mentions protein staining methods and points viewers to a related video on Western blotting for further analysis.

Takeaways

  • 🧬 SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a technique used to separate proteins based on their molecular weight.
  • 🔬 The gel used in SDS-PAGE consists of a stacking gel and a resolving gel, each with different pH levels and pore sizes to facilitate protein separation.
  • 🌡️ The stacking gel has a pH of 6.8 and larger pores with lower ionic strength, while the resolving gel has a pH of 8.8 with smaller pores and higher ionic strength.
  • 🧪 Polyacrylamide gels are made from acrylamide and bisacrylamide, and their ratio can be adjusted to create different pore sizes for protein separation.
  • 🔄 Proteins are denatured with SDS and beta-mercaptoethanol to break disulfide bonds and mask charges, resulting in uniform negatively charged proteins.
  • 🚫 The electrophoresis process involves the movement of proteins from the negative to the positive terminal due to their negative charge from SDS coating.
  • 🏃‍♂️ Small ions like chloride move quickly towards the positive terminal, followed by proteins, with larger, slower glycine ions lagging behind.
  • 🏁 Proteins are 'stacked' at the interface of the stacking and resolving gels before being separated in the resolving gel based on their molecular weight.
  • 🖼️ After electrophoresis, gels can be stained with Coomassie Brilliant Blue or silver stain to visualize protein bands for analysis.
  • 🔍 Western blotting is a further technique used to confirm the presence of a protein of interest in a sample after SDS-PAGE separation.

Q & A

  • What is SDS-PAGE?

    -SDS-PAGE, or SDS polyacrylamide gel electrophoresis, is a technique used to separate proteins based on their molecular weight by utilizing a gel system.

  • What are the two types of gels used in SDS-PAGE?

    -The two types of gels used in SDS-PAGE are the stacking gel, with a pH of 6.8, and the resolving gel, with a pH of 8.8.

  • Why is there a pH difference between the stacking gel and the resolving gel?

    -The pH difference ensures proper separation of proteins. The stacking gel's lower pH helps to 'stack' the proteins at the boundary, while the resolving gel's higher pH facilitates their separation based on molecular weight.

  • What role does pore size play in protein separation in SDS-PAGE?

    -The pore size, which depends on the concentration of acrylamide and bis-acrylamide, affects the separation of proteins. Smaller proteins require higher concentrations of gel to be properly separated.

  • Why is SDS and beta-mercaptoethanol added to the protein solution?

    -SDS denatures proteins and masks their charges with a uniform negative charge, while beta-mercaptoethanol breaks disulfide bonds, ensuring proteins are fully denatured and linearized.

  • How do proteins move during electrophoresis in SDS-PAGE?

    -Since SDS coats proteins with a negative charge, they move towards the positive terminal during electrophoresis, allowing separation based on their molecular weight.

  • What is the role of chloride ions and glycine in the running buffer?

    -Chloride ions, being small and fast, lead the proteins in the electrophoretic process, while glycine ions, which move slowly at pH 6.8, lag behind, aiding in the stacking of proteins in the stacking gel.

  • How are proteins separated in the resolving gel?

    -Once proteins reach the resolving gel, they are separated based on their molecular weight, with larger proteins moving slower than smaller ones.

  • What staining techniques can be used to visualize proteins after SDS-PAGE?

    -Proteins can be stained using techniques like Coomassie Brilliant Blue staining or silver staining to visualize the separated proteins on the gel.

  • What additional step is required to confirm the presence of a specific protein in the sample?

    -To confirm the presence of a specific protein, a Western blot is performed after SDS-PAGE, where the protein of interest is detected using specific antibodies.

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Ähnliche Tags
HTS-PAGEProtein SeparationBiologyMolecular WeightGel ElectrophoresisSDS-PAGEPolyacrylamide GelBiotechnologyLab TechniquesEducational Video
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