Quiz 3

What is the specific goal of the "Red Line" strategy?

• Measures Activity—Testing if the mutant protein is chemically capable of moving lipids, regardless of its location in the fly.

• Environment: In vitro (in a test tube).

• Primary Technique: SDS-PAGE and PI Transfer Assays—You use the bacteria (BL21) as a factory to make a "clean" version of the protein to test its strength.

• What it proves: If the purified protein fails the transfer assay, the mutation has broken the "machinery" of the protein itself.

Why are Sal I and Not I used for the restriction digest?

These enzymes flank the PCR insert site in the $pGEX$ plasmid, allowing the insert to be "liberated" (cut out) for analysis.

Compare DH5alpha vs. BL21 bacterial strains.

• DH5alpha: Optimized for plasmid/insert stability (recA mutation prevents recombination); used for long-term storage.

• BL21: Optimized for high-level protein expression (Protease-deficient to prevent degradation of recombinant proteins).

What is the role of IPTG in the pGEX system?

IPTG acts as an inducer that turns on the Ptac promoter, inducing protein synthesis.

Why use an inducible promoter like Ptac instead of constant expression?

Because some recombinant proteins are toxic to bacteria; induction allows cells to reach a high density before the "switch" is flipped to make protein.

How do you confirm successful protein induction on an SDS-PAGE gel?

You compare pre-induction and post-induction samples; a successful result shows a unique band in the induced lane that is absent in the pre-induction lane.

What is the expected size of the GST-PITP fusion protein?

Between 37-50 kDa (GST tag is ~28 kDa + the PITP domain).

What are the functions of SDS and DTT in the protein sample buffer?

• SDS: Anionic detergent that denatures proteins and provides a uniform negative charge.

• DTT: Reducing agent that breaks disulfide bonds to fully linearize the protein.

What makes up the "Polymerase Gel" and the "TGS Buffer"?

• Gel: 4-15% precast polyacrylamide gradient gels (acrylamide monomers + bis-acrylamide cross-linker).

• TGS Buffer: Tris-Glycine-SDS.

What is the primary goal of the "Green Line"?

• Measures Expression—Testing if the mutant protein is physically present and correctly localized within the living fly eye.

• Environment: In vivo (in the living organism).

• Primary Technique: Immunofluorescence (IF)—Using glowing antibodies to see if the protein is at the subrhabdomeric cisternae

• What it proves: If you see a "glow" under the microscope, the protein is being expressed and sent to the right place.

Why must you "fix" tissue with 2% Paraformaldehyde?

To "freeze" or cross-link proteins in their native cellular positions so they do not move or degrade during the procedure.

Why are white-eyed flies used instead of red-eyed flies for IF?

The red pigment in wild-type flies auto-fluoresces, which would mask the fluorescent signal from the antibodies.

Compare Whole Mounts vs. Cryosectioning.

• Whole Mount: Views protein expression in the entire eye using a confocal microscope to create "z-stacks".

• Cryosectioning: Involves slicing thin sections with a cryostat; it is better for seeing specific cellular layers.

Where is the rdgB protein localized in a wild-type retina?

The subrhabdomeric cisternae, where it is critical for membrane transport.

Problem: No protein expression on gel.

Check: Did you add IPTG? Is there a frame-shift mutation in your vector?

Problem: Protein is insoluble (inclusion bodies).

Solution: Lower the induction temperature or the concentration of IPTG

Problem: Uncut plasmid shows multiple bands.

Forms: Supercoiled (fastest), Linear (middle), and Nicked/Relaxed (slowest).