Lab 9

Question 1

Physics of fluorescence

Answer 1

• Electron in molecules that produce fluorescence move ot higher energy orbital as a result of excitation
• Excited electrons often go back to initial ground state - and release excess energy in the form of light (emission)

Question 2

Fluorophores and properties of excitation

Answer 2

• Fluorophores are molecules with aromatics with conjugated bonds
• More conjugated bonds - lower light energy needed to excite them
• Fluorophores have characteristic wavelengths for excitation and emission

Question 3

What is original GFP?

Answer 3

Discosoma sp
(DsRed)

Question 4

Functional advantage of fluorescence

Answer 4

• In coral - protects endosymbiotic dinoflagellates from excessive sunlight
• Antioxidant by quenching superoxide radicals
• Regulation of endosymbiotic dinoflagellates
• Interspecies communication in fish
• Camouflage

Question 5

GFP structure

Answer 5

• Barrel with 11 beta sheets and central helix containing chromophore
• Barrel interioir is crowded with amino acid residue side chains
• Alpha-helices at ends of protein stabilize it
• Chromophore is formed by post-translational modification that result in cyclization of peptide backbone

Question 6

Which residue generates chromophore?

Answer 6

Residue 65-67

Question 7

Chromophore structure

Answer 7

• Backbone cyclization generates a 2-ring structure
• Maturation
• Planar structure
• Other amino acid residues in structure are important to catalyze this change

Question 8

Residues that affect properties in GFP

Answer 8

• Residue substitution in chromophore can lead to colour change
• Substitution of residues in beta barrel close to chromophore may also change colour

Question 9

Fluorescent highlighters

Answer 9

• Structure of chromophore - can change in different lights in special FPs
• Change can result in photoconversion
• Change can be reversible or irreversible

Question 10

FP properties for practical use

Answer 10

• Brightness
• Maturation Rate
• Photostability
• Oligomeric nature and aggregation
• pHstability

Question 11

mNeonGreen original protein

Answer 11

LanYFP is structurally similar to GFP, but has a very different sequence
- “brightest natural FP known”
- Mutagenized to be monomeric

Question 12

Gene fusion of protein of interest

Answer 12

Cloning before introducing to the organism with a vector
or homologous recombination

Question 13

3 steps of todays experiment

Answer 13

1. Reactivate membrane
2. Verify efficiency of transfer using stain-free techno
3. Probe membrane with specific antibodies

Question 14

What is used to probe protein?

Answer 14

• Use a “primary antibody” - an antibody that binds specifically to protein of interest or affinity tag on protein

Question 15

What is “blocking”?

Answer 15

A step needed because membranes have high capactity for protein binding
- Incubate membrane with proteins that won’t interfere with antibody detection, like milk proteins - non-specific binding of antibodies to membrane can be avoided
- Reduces background signal from non-specific binding

Question 16

Why use two antibodies?

Answer 16

Sigal amplification by secondary antibody and allows primary antibody to be used for diverse applications in the lab
- primary is specific for the POI
- secondary is specific to animal species POI was raised in

Question 16

3 parts of experiment

Answer 16

1. Blocking and primary antibody incubation
2. Secondary antibody incubation and detection
3. Analysis

Question 16

What is a secondary antibody?

Answer 16

Secondary antibody conjugated to a detection agent is used (HRP) - attaches to primary and is visible (primary isn’t visible on its own)