Gps explorer 3

The appropriate band identified to bind with LBD1 was excised from SDS-PAGE gel, placed into a clean microcentrifuge tube, and then digested with trypsin as described [47 (link)]. The digested band was run on a MALDI-TOF/TOF 5800 mass spectrometry (AB SCIEX, USA) to obtain the corresponding peptide mass fingerprinting (PMF) using GPS Explorer 3.5 (Applied Biosystems, USA) and Mascot (Matrix Science, UK). PMF data were further indexed using Andromeda search engine to assign the corresponding peptide sequences in an integrated silkworm proteome database [48 (link), 49 ].

The recombinant strain BW (pBAD-ectABC) was grown at 37°C in LB broth with ampicillin. When the optical density of the culture at 600 nm reached 0.6, L-arabinose was added at a final concentration of 0.1% and incubated at 30°C for 6 h. The cells were harvested by centrifugation and resuspended in 50 mM of potassium phosphate buffer (pH 7.0). The cell suspension was sonicated (Soniprep 150 sonifier) and centrifuged (12,000 × g, 10 min). The supernatant was subjected to SDS-PAGE analysis. For mass spectrometric protein identification, bands of interest were excised and submitted to in-gel reduction, alkylation, and digestion with bovine trypsin (12.5 ng/μl, sequencing grade; Roche Applied Science) as previously described by Sechi and Chait [30 (link)]. Afterward, the trypsinized peptides were analysed on an Applied Biosystems 4700 Proteomic Analyzer (Applied Biosystems, Framingham, MA) as previously described [31 (link)]. The resulting MS and MS/MS data were analyzed and peak lists were generated using GPS Explorer 3.5 (Applied Biosystems) and were further searched using MASCOT 2.0 search engine (MatrixScience, London, U.K.) against SwissProt protein sequence database download in Dec, 2014 from NCBI.