Section 3: Isolating organelles and molecules

Question 1

What surface proteins do T cells have that can receive fluorescent antibodies?

Answer 1

Thy1.2 (green) and CD3 (red)

Question 2

What is the purpose of FACS and how does it work?

Answer 2

FACS (fluorescent activated cell sorting) sorts and counts labelled, live cells (ex., T cells)

1. Cells pass singloe file through a light beam
2. Individual cells are given a charge proportional to the degree of fluorescence detected by the detectors
3. Cells are separated by an electric field and collected, sort, and counted

Question 3

The first step in isolating cell organelles is disrupting the plasma membrane of the cell. Describe 5 ways this can be done

Answer 3

1. Mechanical homogenization (blender)
2. Sonication (vibrates and lyses cells at controlled intensities)
3. Pressure (cells are forced through a very narrow valve/needle)
4. Non-ionic detergents (ex., Triton X100)
   
   • Must only be applied in a specific amount for a specific duration of time
5. Hypotonic solutions (water rushes into the cells; lyses them)

All of these create a “cell homogenate” (mush)

Question 4

The second step in isolating cell organelles is centriguation of the cell homogenate. Describe the differential centriguation method

Answer 4

• Spinning homogenate yields pellet and supernatant
• Centrifugal force is increased to isolate specific organelles based on their mass (sequentially remove things we don’t want)

Question 5

The second step in isolating cell organelles is centriguation of the cell homogenate. Describe the equilibrium density-gradient centrifugation method

Answer 5

• Separation based on density
• Homogenate is applied to a gradient of sucrose
  
  • At high speeds, organelles migrate to the sucrose layer equal to their own density and remain there

Question 6

How are proteins separated from organelles?

Answer 6

• Strong/ionic detergents are used
  
  • Non-ionic detergents disrupt lipid bilayers
  • Ionic detergents disrupt ionic and hydrogen bonds and denatures proteins in addition to lysing all membranes
    
    • This is due to their ionic group
• Detergents are amphipathic molecules (hydrophobic and hydrophilic)

Question 7

Describe the process of SDS-PAGE

Answer 7

• Electrophoretic separation of proteins is performed in polyacrylamide gels (PAGE - polyacrylamide gel electrophoresis)
• Carried out in the presence of negatively charged SDS detergent
  
  • Binds to (coats) and destabilizes the hydrophobic side chains in proteins
    
    • Gives them a negative charge proportional to their size
• Coomassie brilliant blue and silver dye can be used to visualise the gel
  
  • Low mass proteins have greater mobility than high mass proteins

Question 8

How can a specific protein be identified from the rest?

Answer 8

Antibodies can recognise the desired protein which has been tagged with antigens

Enzyme labelled secondary antibodies attach to the primary antibodies

An enhanced chemiluminescent substrate reacts with the enzyme on the secondary antibody and creates fluorescence

X-ray film or digital imaging can detect this