lab 4

• What does SDS-PAGE separate proteins by?

  Molecular weight

  
• What is the process of separating macromolecules in an electric field called?

  Electrophoresis

  
• What is used as a support medium in SDS-PAGE?

  Discontinuous polyacrylamide gel
• What does SDS do to proteins in SDS-PAGE?

  Denatures and coats with negative charge

  
• How do larger molecules behave in polyacrylamide gels?

  They are restrained from migrating
• What primarily determines the final separation of proteins in SDS-PAGE?

  Differences in relative molecular mass
• What happens to proteins with the same molecular weight in an electric field?

  They migrate at different speeds
• What is the purpose of reducing agents like DTT in SDS-PAGE?

  To break down disulfide bonds
• What is the role of the running buffer in SDS-PAGE?

  To conduct current through the gel
• What are the main components required for SDS-PAGE gel preparation?

  • 1.5M Tris-HCl pH:8.8
  • 1M Tris-HCl pH:6.8
  • 10% SDS
  • 30% Acrylamide
  • 10% APS
  • TEMED
  • Distilled water
  • Gel combs
  • Casting stands
  • Glass plates
• What is the purpose of the protein ladder in SDS-PAGE?

  To provide molecular weight standards

  
• What is the first step in preparing the separating gel for SDS-PAGE?


  Mixing components in a Falcon tube
• What should be done immediately after adding TEMED to the gel mixture?

  Mix and transfer the mixture immediately
• Why should TEMED be opened only inside the hood?

  It can cause severe digestive tract irritation
• What temperature should samples be boiled at to denature proteins?

  90
• What is the purpose of the Laemmli Sample Buffer?

  To denature proteins and add negative charge
• What happens to proteins during the boiling step?

  They are completely denatured
• What must be done before removing gels after electrophoresis?

  Turn off the power and remove cables
• How long should the gels be run at 120 volts?

  Approximately 40 minutes
• What is the purpose of Coomassie Blue staining solution?

  To visualize proteins in the gel
• How long should the gel be stained with Coomassie Blue?

  10 minutes
• What should be done after staining the gel with Coomassie Blue?

  Rinse with deionized water
• What is the purpose of the destaining solution?

  To remove excess Coomassie Blue stain
• How long should the gel be incubated in the destaining solution?

  10 minutes
• What should be done with the gel after destaining?

  Add deionized water overnight
• Who should take a picture of the gel the next morning?

  One member from each group