🧪 Method Theory – Complete & Educational Summary

This chapter introduces key molecular biology and imaging methods used to study proteins, cells, and tissues—especially in C. elegans. Each method explains what it is, how it works, and why it is used.

🟢 4.0.1 Green Fluorescent Protein (GFP)

What is GFP? Green Fluorescent Protein (GFP) is a naturally fluorescent protein originally isolated in the 1960s from the jellyfish Aequorea victoria. It is widely used as a fluorescent marker in molecular and cell biology.

Key properties

• Excited by blue light (≈395 or 475 nm)
• Emits green light (≈510 nm)
• Does not require enzymes or cofactors → works inside living cells

Structure

• 238 amino acids
• Barrel-shaped structure:
  • 11 β-sheets forming a protective barrel
  • Central α-helix
• Contains the p-hydroxybenzylidene-imidazolinone (p-HBDI) chromophore, which is responsible for fluorescence

Why GFP is so useful

• Can be fused to proteins at:
  • N-terminus
  • C-terminus
  • Internal positions
• GFP-tagged proteins usually retain their native function
• Enables real-time visualization of protein expression and localization

🧠 Key idea: GFP turns invisible biological processes into visible green signals.

🐛 4.0.2 GFP Reporter Strains in C. elegans

Why C. elegans?

• Small size
• Transparent body
• Easy genetic manipulation ➡️ First multicellular organism where GFP was used successfully

Advantages of GFP reporters

• Live imaging (no fixation required)
• No behavioral disturbance
• Superior to older reporters like lacZ

Two types of GFP reporter constructs:

1️⃣ Transcriptional reporters

• GFP gene fused to the promoter of a gene of interest
• Reports where and when transcription occurs
• Does not show protein localization

2️⃣ Translational reporters

• GFP fused directly to the gene of interest
• Under the gene’s native promoter
• Reports where the protein is located in the cell

🧠 Key distinction:

• Transcriptional = where gene is ON
• Translational = where protein is

⚡ 4.0.3 SDS-PAGE (Protein Separation)

What is SDS-PAGE? Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is used to separate proteins by molecular weight.

How it works

• SDS is an anionic detergent
• It:
  • Denatures proteins (destroys secondary & tertiary structure)
  • Coats proteins with negative charge
• Creates a constant charge-to-mass ratio for all proteins

➡️ Result: separation depends only on protein size

Migration principle

• Proteins move toward the anode (+)
• Smaller proteins migrate faster and further through the gel

Reducing agents

• Commonly DTT (dithiothreitol)
• Breaks disulfide bonds
• Improves accuracy of size-based separation

🧠 Key idea: SDS-PAGE makes all proteins “equal” except for size.

🧬 4.0.4 Western Blot

Purpose Western blot is used to detect and quantify specific proteins using antibodies.

Main steps

1. Protein separation by SDS-PAGE
2. Transfer of proteins to a nitrocellulose membrane
3. Detection using antibodies

Antibodies

• Primary antibody
  • Binds directly to the protein of interest
  • Commonly raised in mouse or rabbit
• Secondary antibody
  • Binds the Fc region of the primary antibody
  • Conjugated to enzymes like HRP (horseradish peroxidase)

Detection

• HRP catalyzes a reaction that produces chemiluminescence
• Light emission reveals protein presence and size

Blocking step

• Uses skim milk + buffer + detergent
• Prevents non-specific antibody binding

Normalization

• Required due to loading variability
• Uses housekeeping proteins as loading controls:
  • GAPDH
  • β-actin
  • α-tubulin

Stripping

• Antibodies can be removed from the membrane
• Allows reuse of the same membrane for multiple proteins

🧠 Key idea: Western blot answers “Is my protein there, and how much?”

🧵 4.0.5 Phalloidin Staining (Actin Visualization)

What is phalloidin?

• A toxic cyclic peptide (phallotoxin)
• Derived from Amanita phalloides (death cap mushroom ☠️)

What does it bind?

• Specifically binds F-actin (filamentous actin)
• Does not bind G-actin (monomeric form)

Why the binding is specific

• During polymerization:
  • Actin subunits flatten
  • A cleft opens between adjacent subunits
• Phalloidin binds in this cleft and stabilizes F-actin

Why it’s important

• Prevents depolymerization
• Preserves cytoskeletal structure
• Considered a gold standard for actin staining
• Commonly used for muscle fibers in C. elegans

Fluorescent labeling

• Phalloidin itself is not fluorescent
• Conjugated to dyes (commonly rhodamine)

TRITC-phalloidin

• Uses tetramethylrhodamine isothiocyanate
• Red-orange fluorescence
• High resistance to photobleaching
• Does not interfere with actin binding

🧠 Key idea: Phalloidin “locks” actin filaments in place so we can see them.

🔬 4.0.6 Spinning Disk Confocal Microscopy (SDCM)

What is SDCM? A fluorescence imaging technique that produces high-resolution images with low background noise.

Core principle

• Uses a spinning disk with many pinholes
• Only in-focus light passes through pinholes
• Out-of-focus light is blocked

Key components

• Monochromatic laser (excitation)
• Microlens disk (focuses incoming light)
• Pinhole disk (filters out-of-focus light)
• Dichroic mirror (directs wavelengths)
• Objective lens (magnification + resolution)
• Barrier filter (selects emission wavelength)
• Sensor/camera (image detection)

Why spinning matters

• Disk spins rapidly in an Archimedean spiral
• Allows simultaneous scanning of many points
• Produces images quickly with minimal phototoxicity

Advantages

• Fast imaging
• High signal-to-noise ratio
• Ideal for live samples and fluorescent reporters

🧠 Key idea: SDCM sees only what’s in focus—fast and gently.

🧬 4.0.7 Mutation of C. elegans

Why mutants matter

• Essential for linking genes → phenotype
• Enable functional genetic studies

DNA modification approaches

• Some methods are non-targeted
• A common example: EMS mutagenesis

EMS (Ethyl Methanesulfonate)

• Acts on germline cells
• Mutation rate: ~2.5 × 10⁻³ mutations per gene per generation
• Mechanism:
  • Adds ethyl group to guanine
  • Forms O⁶-ethylguanine
  • Alters base pairing (guanine pairs with thymine)
  • Results in point mutations

After mutagenesis

1. Screen for desired phenotype
2. Isolate mutant
3. Backcross 5 times with wild-type males
   • Reduces unwanted background mutations
   • Retains the mutation of interest

🧠 Key idea: EMS creates random mutations; careful screening and backcrossing make them useful.

✅ Big Picture Take-Home Message

This chapter provides the methodological foundation for:

• Visualizing proteins (GFP, microscopy)
• Separating and detecting proteins (SDS-PAGE, Western blot)
• Studying cytoskeletal structure (phalloidin)
• Imaging with high precision (SDCM)
• Linking genes to function (mutant C. elegans)

Together, these methods allow mechanistic insights from gene → protein → structure → phenotype.