Juvenile Hormone Affinity Purification

Drosophila S2 cell frozen cells were pelleted by centrifugation and prepared by glass Dounce homogenizing on ice in lysis buffer (I-Per insect protein extraction reagent; Fisher Scientific) containing Halt™ protease inhibitor cocktail with EDTA (Fisher Scientific). Cell lysates were centrifuged 13.2k x g for 20 min at 4°C and the supernatant was collected and used as the initial whole cell lysate. For the JH affinity column, 10 cell pellets were homogenized and the whole cell lysate was recirculated through the control linoleate column for 1 hour at 4°C to remove non-specific binding proteins; the flow-through was transferred to the JH affinity column and recirculated for 4 hours at 4°C. The columns were washed with 20 column volumes of bead buffer (50 mM Tris pH 6.8, 5 mM sodium fluoride, 240 mM sodium chloride, 100 µM benzamidine HCL 5mM EDTA, 5 mM EGTA, 0.1% triton X-100, Halt™ protease inhibitor cocktail) until the OD280 fell below 0.05. The JH affinity column was eluted by recirculating bead buffer plus 10 mg/ml methoprene for 4 hours at 4°C. The control column was eluted by adding release buffer (10% sodium dodecyl sulfate (SDS), 1% beta-mercaptoethanol) and the resin brought to 95°C. The resin was centrifuged and the supernatant collected. The process was repeated several times and the supernatants pooled. Eluted proteins were concentrated against 20% polyethylene glycol in SDS reservoir buffer, separated on Precise™ 4-20% Tris-HEPES Gels (Fisher Scientific) and visualized by non-destructive silver staining (Blum et al. 1987). Protein gels from the control and JH resins were compared; bands unique to the JH resin were identified, cut from the gel and taken to the University of Wisconsin Biotechnology Center for tryptic digest and identification by mass spectrometry. Synthesis and elution for the methoprene and linoleic affinity columns followed the same procedures but cell lysates were eluted with 8M urea, concentrated with a 10K spin column (Amicon) and exchanged to 500mM ammonium bicarbonate before the sample was sent to University of Wisconsin Biotechnology Center for tryptic digest and identification by mass spectrometry.