Oasis u hlb plates

Ovaries were dissected from mature adult female flies in Grace’s insect media, and the individual follicles were separated with forceps. Samples were then collected at 300 oocytes/sample. Follicle cells were removed from stage 14 oocyte as described above. Mitochondria were isolated as described above, and protein was precipitated via TCA. Mitochondrial protein was constituted in 500 mM TEAB( + 1%SDS), and 100 ug was taken for processing and analysis.
Proteins in solution (100 ug in 50 ul) were reduced with DTT (x3) and then alkylated with iodoacetomide (x3). 15 ul (30 ug) of the protein solution was precipitated the remainder was frozen. The TCA/acetone pellet was reconstituted with 20ul 500mmTEAB(+10 uL water) and sonicated for 10 min. The sonicated solution was then proteolyzed with trypsin (frozen, Promega) 20 ng/ul. Following digestion, peptides were desalted on Oasis u-HLB plates (Waters). The digested peptides were reconstituted in 8 ul (2%ACN/0.1%FA). 1 ul (12% of the sample) was analyzed by LC/MSMS on a nano- LC ESI LTQ_Orbitrap_Velos (ThermoFisher) in FTFT using 90 min total gradient, resolution at 400 Da 60 K for Full MS and 15 K for MS2. The injected peptides showed an abundant (∼1 uG) base peak chromatograph.

Strains expressing the ApdA reporter were grown in 100 mL of LB with 20 μM bortezomib until OD₆₀₀ = 0.5 and harvested by centrifugation. The pellet was frozen at −80 °C and thawed in 2× CellLytic B-cell lysis reagent (Sigma) and 0.2 mg mL⁻¹ lysozyme for 10 min. The lysate was clarified by centrifugation for 30 min at 20,000×g at 4 °C. In total, 50 μL of Strep-tactin Sepharose beads (IBA) were added to the supernatant and samples were incubated at 4 C for 1 h. The beads were washed four times with IP wash buffer (20 mM Tris pH 8.0, 100 mM NH₄Cl, 0.4% Triton X-100, 0.1% NP-40) for 5 min at 4 °C. Protein was eluted from the beads by shaking at 4 °C in elution buffer (20 mM Tris pH 8.0, 100 mM NH₄Cl, 5 mM desthiobiotin) for 1 h. Then, 36 μl of the immunoprecipitated sample was reduced with 100 mM DTT in 100 mM triethylammonium bicarbonate (TEAB) buffer at 58 °C for 55 min and then the pH was adjusted to 8.0. The samples were alkylated with 200 mM iodoacetamide in 100 mM TEAB buffer in the dark at room temperature for 15 min. Proteins were pelleted and resuspended in 50 mM TEAB and proteolyzed with 15 ng μL⁻¹ of LysC (Wyco) at 37 °C overnight. Peptides were desalted on Oasis u-HLB plates (Waters), eluted with 60% acetonitrile (ACN)/0.1% trifluoracetic acid (TFA), dried and reconstituted with 2% ACN/0.1% formic acid.