L3 - Isolation and analysis of cell organelles and molecules

Fluorescent antibodies or stains:

• labeling %%live%% cells,
  • Proportion of staining = proportion of DNA,
  • Fluorophore = fluorochrome.
• 
• Antibodies:
  • Fluorophores bounds to antibodies against specific cell surface proteins
• Membrane Permeable Dyes:
  • Membrane permeable fluorescent dyes labels intracellular structures
  • (i.e. Hoechst stain binds DNA in nucleus).
• Cells with bound antibodies or that have taken up the dyes can then be sorted and counted.

Fluorescent Activated Cell Sorting (FACS):

1. Cells pass $single file$ through a laser light beam in a thin tube,
2. Both fluorescent light emitted and scattered are measured by detectors,
3. Individual cells are forced through a nozzle and given a $charge proportional to the degree of fluorescence detected$,
4. Cells with different electric charges are $separated by an electric field$ and collected.

• Many different types fluorophores can be used to differentiate different cells

Quantification of Cells Expressing 2 Different Cell Surface Markers by FACS:

• As the cells pass through the FACS machine, the intensity of the green and red fluorescence emitted by each cell is recorded
• Each dot represents a single cell
• The proportion of each cell population can be calculated.

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Cell Cycle Analysis by FACS:

• Cells that have replicated their DNA but not fully divided (G2) will have twice the Hoechst stain fluorescence intensity of non-dividing cells (G1).
• 

Organelles of Cells:

 Cell Organelle Isolation:

• STEP ONE: DISRUPTION of CELL PLASMA MEMBRANE
  • i) mechanical homogenization
  • ii) sonication  (ultrasound) (destroys the membrane)
  • iii) pressure (cells are forced through a very narrow valve) (cause the cell to rupture)
  • iv) non-ionic detergents i.e.,  Triton X-100
  • Agent that causes cell membranes to dissipate
  • disperses phospholipid bilayer
  • v) placing cells in hypotonic solution
  • Osmotic effect that makes cell blow up (swollen)
• STEP TWO: CENTRIFUGATION of CELL HOMOGENATE
  • i) differential
  • ii) equilibrium density-gradient.

Centrifugation:

• Refrigeration:
  • Cools down the system to prevent the samples from overheating
• Vacuum
  • Prevents heat conduction
• Armoured chamber:
  • Prevents the materials from leaving the system in a projectile motion
• Distribution
  • equal weight, even distribution, balance the system to prevent shaking \n

• Differential Centrifugation:
  • spinning homogenate yields pellet & supernatant
  • increasing centrifugal force (gravity) to isolate organelles based on mass
  • larger organelles = easy to separate, low force required
  • smaller organelles = need high speed to separate
  • important to do it sequentially
    • big, medium, small.
  • g = Relative centrifugal force (RCF).
  • Relative g required to separate organelles:
  • 600g * 10 min
    • Nuclei
  • 15,000g * 5 min
    • mitochondria, chloroplasts, lysosomes, and peroxisomes
  • 100,000g * 60 min
    • plasma membrane, microsomal fraction(framents of endoplasmic reticulum) and large polyribosomes
  • 300,000g * 2h
    • ribosomal subunits, small polyribosomes
  • Pour out
    • soluble part of cytoplasm (cytocol)
• 
• Equilibrium Density-Gradient Centrifugation:
  • separation based on density
  • homogenate is applied to a gradient of sucrose
  • at high speed/several hours, organelles migrate to sucrose layer equal their own density and remain there.
  • each layer then can be suck up for later use
  • 

Separating Protein from Organelles:

• Detergents:
  • $amphipathic$ molecules.
  • non-ionic detergents:
  • only disrupts lipid bilayer.
  • ionic detergents:
    • $disrupts lipid bilayer and proteins$
    • by denaturing ionic & H-bonds that breaks down proteins.
• 

SDS-PAGE:

• $**Polyacrylamide gel electrophoresis (PAGE)**.$
  • Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels.
  • usually carried out in the presence of the ^^negatively charged detergent SDS^^ and called $SDS-PAGE.$
• $SDS Denaturing$
  • SDS binds to and destabilizes the hydrophobic side chains within the core of proteins
  • All polypeptide chains are forced into extended negatively charged conformations with a @@similar charge-to-mass ratio@@.
  • The mobility of the SDS-protein complexes are influenced primarily by molecular size, i.e MW=daltons
• 
• 

Quantification and Observation of Protein:

• Chemical dyes allow the visualization of protein gradients

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• {{There is a linear relationship between the @@log MW@@ and the electrophoretic mobility.{{
• Low mass proteins have greater mobility than high mass proteins
• 

Western Blotting (Immunoblotting):

• Very useful in separating proteins as many proteins have similar/same molecular mass

Procedure:

1. Electrophoresis and transfer

• Electric current helps SDS-polyacrylamide gel to separate the proteins

1. Antibody detections

• Incubate with antibody1 that recognizes the desired protein
  • Ab 1 recognizes the desired protein
• Incubate with enzyme linked antibody2
  • Ab2 recognizes Ab1
• Enzyme-labelled antibody2 recognizes the antibody1
  • wash excess after procedure

1. Chromogenic detection

• React with substrate for antibody2 -linked enzymes
  • Enhanced chemiluminescence substrate reacts with enzyme in Ab2 (glows)
  • luminal reacts with the enzyme product and emits light
  • imaging with x-ray detector or very sensitive camera
    • forms a bright band
    • sometimes inverted to be a dark band

Immunoblotting to detect changes in levels of a specific protein:

• Loading control: actin
  • Does not change by the amount of reactant
  • Tests if the gel was applied properly
  • if actin band changes width/intensity, then there’s excess gel

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