The blot was blocked with 10% skim milk solution for 2 hours. After washing with phosphate-buffered saline (PBS) solution, the blot was probed overnight using a PD0325901 polyclonal flagellar antibody raised in a rabbit against isolated flagellar filaments [41]. Protein A-alkaline phosphatase (Sigma-Aldrich) was used as the secondary antibody. The blot was washed with PBS and was developed using NBT/BCIP (Sigma). Preparation of samples for tandem mass spectrometry analysis (MS/MS) The flagellar protein samples were run on a polyacrylamide gel as described above. Staining and destaining of the protein gel were performed following standard protocols
[42]. The gel was soaked overnight in a staining solution containing 0.1% Coomassie Brilliant Blue (R-250; Sigma), 40% methanol, and 10% acetic acid. Destaining was done using a solution containing 40% methanol and 10% acetic
acid. The bands (between approximately 25-37kDa) see more were excised and submitted to the Southern Alberta Mass Spectrometry (SAMS) Centre at the University of Calgary Selleckchem TPX-0005 for LC-MS/MS analysis. Two bands within the size range were observed in the gel. The two bands were analyzed separately for 3841 and in combination for VF39SM. The gel slices were rinsed once with HPLC-grade water and then twice with 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile. The gel slices were dehydrated with acetonitrile prior to lyophilization. The dehydrated gel was resuspended in 25 mM ammonium bicarbonate (pH8.0) and samples were digested with trypsin. The peptides were extracted from the gel using 1% formic acid in 50% acetonitrile. The extracts were reduced to dryness and then reconstituted in mobile phase of the buffer (3% acetonitrile with 0.2% formic acid) for liquid chromatography. Tandem mass spectrometry analysis (MS/MS) The digests were analyzed using
an integrated Agilent 1100 LC-Ion-Trap-XCT-Ultra system (Agilent Technologies, Santa Clara, CA), which has an integrated Lumacaftor clinical trial fluidic cartridge for peptide capture, separation, and nano-spraying (HPLC Chip). The injected samples were trapped and desalted for 5 minutes using a pre-column channel (40-nl volume; Zorbax 300 SB-C18) with an auxiliary pump that delivers 3% acetonitrile and 0.2% formic acid at a flowrate of 4 μl/minute. The peptides were reverse-eluted from the trapping column and separated on a 150 mm-long analytical column (Zorbax 300SB-C18) at a flowrate of 0.3 μl/minute. The peptides were eluted using a 5-70% (v/v) acetonitrile gradient in 0.2% (v/v) formic acid over a period of 10 minutes. The MS/MS spectra were collected by data-dependent acquisition, with parent ion scans of 8100 Th/s over m/z 400-2,000. MS/MS scans at the same rate over m/z 100-2200. Mass Spectrometry Data Analysis DataAnalysis software for the 6300 series ion trap, v3.4 (build 175) was used to extract the peak-list data. The MS/MS data were analyzed using Mascot v2.