Part 2: L3, Organelle Targeting Fluorophores

Question 1

Key features for organelle targeting:

Answer 1

• Membranes and thus membrane potentials (+ or -)
• Charge
• Lipophicility
• Other biochemistry
• (Antibody )

Question 2

Give 2 minor groove DNA binding stain examples:

Answer 2

DAPI:
* Minor groove binder
* Binds to A-T rich regions of DNA
* Protonated at physiological pH (cationic)
Hoescht:
* Protonated (cationic)

Question 3

DNA intercalator example:

Answer 3

• Acridine orange
• Intercalates (i.e. Pi stacks) DNA (glows green), binds to RNA electrostatically (glows orange)

Question 4

Nuclear Imaging Agents Overview:

Answer 4

• Cationic (attracted to DNA phosphate)
• Lipophilic (cross internal membrance)
• Groove binders or DNA intercalators)
• Usually planar heterocycles

Question 5

Mitochondria crucial features:

Answer 5

• Complex internal structure
• Multiple membranes
• Membrane potential is 10x mroe negative than cytoplasm
  -> cationic lipophilic agents taken in well

Question 6

Membrane stains (qualities)

Answer 6

• Highly lipophilicity (C12-C18)
• Chains simply attrcated to membranes by lipophilicity / hydrophobicity

Question 7

Protein Tagging dyes: basic structure

Answer 7

• N-terminus = amine reactive
• C-terminus = carboxylate reactive (hard in water)

Question 8

Antibody tagging

Answer 8

• Labelling organelles by tagging anitibodies which bind to specific target antigens produced in the cell then purified

Question 9

GFP: Structure, use and variants

Answer 9

• GFP fluorophore is a simple conjugated system but lives in a 238 amino acid peptide
• Commonly genetically engineered into other peptides, organisms etc
• Variations include CFP (cyan FP), RFP (red FP) etc.

Question 10

What is costaining?

Answer 10

• Validating a new agent by co-staining with a knwon agent that emits / absorbs a different wavelength, then acquire 2 images and overlay