Equipment and Materials

Week 1

• T-100 Thermal Cycler (BioRad USA, Hercules, CA)
• Drosophila melanogaster larval culture
• EBR (0.129 M NaCl, 4 mM KCl, 2 mM CaCl₂, 35 mM Tris-HCl pH 6.85)
• 70% ethanol
• Plastic pestle fitted to the 1.5 ml microcentrifuge tube
• Bovine calf serum (BCS) (KC Biological, Lenexa, KS), fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO)
• 3X SDS Laemmli sample buffer (187.5 mM Tris pH 6.8, 6% sodium dodecyl sulfate [SDS], 30% glycerol, 15% β−mercaptoethanol [βME], 0.015% bromophenol blue [BPB])
• 1.5 ml microcentrifuge tube
• 0.2 ml PCR tube
• Quick Start™ BioRad protein assay (Bio-Rad, Hercules, CA)
• 13×100 mm glass tubes

Week 2

• Bio-Rad Mini-PROTEAN II Cell (Bio-Rad, Hercules, CA)
• Mini-Trans-Blot Cell (Bio-Rad, Hercules, CA)
• 4–20% SDS-Tris HCl gels or 10% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA )
• Tris-glycine running buffer (0.025 M Tris-base, 0.192 M glycine, 0.1% SDS)
• Precision Plus Protein Standards (Bio-Rad, Hercules, CA)
• PVDF membrane (Bio-Rad, Hercules, CA)
• Blotting buffer (0.025 M Tris-base, 0.192 M glycine, 20% methanol, pH 8.3)
• Ponceau S stain (0.2% Ponceau S, 3% TCA, 3% sulfosalicylic acid)
• GelCode™ Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA)

Week 3

• Washing buffer: 1X PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4), 0.1% Triton X-100
• Blocking buffer: 10% bacto-peptone in 1X PBS washing buffer
• Antibody buffer: 1% bacto-peptone in 1X PBS, 0.1% Triton X-100
• Rabbit anti-bovine serum albumin Ab (1.5 mg/ml) (Mp Biomedical, Irvine, CA )
• Alkaline phosphatase-conjugated goat anti-rabbit IgG Ab (MilliporeSigma, Burlington, MA)
• One tablet of BCIP/NBT (Roche Applied Science, Madison, WI) per 10 ml reverse osmosis (RO) water to prepare a ready-to-use alkaline phosphatase substrate solution (0.4 mg/ml NBT; 0.19 mg/ml BCIP; 100 mM Tris buffer, pH 9.5; 50 mM MgSO4 (pH 9.5))