Selected article for: "cell lysate and final volume"

Author: Courtney R. Sullivan; Catharine A. Mielnik; Sinead M. O’Donovan; Adam J. Funk; Eduard Bentea; Erica A.K. DePasquale; Zhexing Wen; Vahram Haroutunian; Pavel Katsel; Amy J. Ramsey; Jarek Meller; Robert E. McCullumsmith
Title: Connectivity analyses of bioenergetic changes in schizophrenia: Identification of novel treatments
  • Document date: 2018_6_5
  • ID: ltb6l5xz_111
    Snippet: For each sample, 1000ul of 10mg/ml antibody coupled beads were washed 2x 1ml with ice cold 1x PBST (#9809S, Cell Signaling) then incubated with mouse brain lysate brought to a final volume of 1000ul with ice cold 1x PBST for 1 hour at room temperature. The supernatant was removed and the beads were washed 4 x 10 minutes at room temperature in 1ml ice cold 1x PBST. Captured protein complexes were eluted with 30ul of 1N Ammonium Hydroxide (#320145,.....
    Document: For each sample, 1000ul of 10mg/ml antibody coupled beads were washed 2x 1ml with ice cold 1x PBST (#9809S, Cell Signaling) then incubated with mouse brain lysate brought to a final volume of 1000ul with ice cold 1x PBST for 1 hour at room temperature. The supernatant was removed and the beads were washed 4 x 10 minutes at room temperature in 1ml ice cold 1x PBST. Captured protein complexes were eluted with 30ul of 1N Ammonium Hydroxide (#320145, Sigma), 5mM EDTA, pH 12 for 10 minutes at room temperature. 6ul of 6x protein denaturing buffer (4.5% SDS, 15% β-mercaptoethanol, 0.018% bromophenol blue, and 36% glycerol in 170 mM Tris-HCl, pH 6.8) was added to each sample elution. The eluted samples were heated at 70°C for 10 minutes then processed for mass spectrometry.

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