Las 3000 intelligent dark box

The transcriptional fusion of flaA (HPP12_0609) to the promoterless gfpmut3 gene in pTM117 (accession number EF540942)^{33 (link)} was constructed by amplification of the promoter region of the flaA gene from P12 genomic DNA using primers FlaA_SacII_F (5′-TCCccgcggGAGCTAAATGCTTGGATATATCCAGCAAT-3′) and FlaA_BamHI_R (5′-CGCggatccCATTTTGAGTGAGTGCGGATTGC-3′) to generate a 1136-bp amplicon. This product was cloned into pGEM-T Easy to generate pGEMflaA, and confirmed by sequencing. The flaA fragment was excised by SacII/BamHI digestion and cloned into the same sites of the transcriptional fusion vector pTM117 to create pTM117-flaA. The pTM117-flaA plasmid was moved into H. pylori strain 7.13 wild-type by natural transformation, and transformants were selected on GC plates containing 10 μg/ml kanamycin. Transformants were graded as GFP-fluorescent or non-fluorescent by their fluorescent intensity detected using LAS-3000 Intelligent Darkbox (FujiFilm) in comparison to H. pylori 7.13 wild-type. Sequence integrity was confirmed by Sanger sequencing for pTM117-flaA plasmid recovered from three GFP-fluorescent and three non-fluorescent transformants; Southern hybridisation analysis of whole genomic DNA confirmed that the flaA-gfpmut3 fusion was retained in the plasmid and had not integrated into the chromosome of 7.13 transformants.

LPS microextraction was prepared as previously described [51 (link)]. Briefly, bacterial cells (OD₆₀₀ = 3.0) harvested from CBA plates were suspended in 100 μL LPS lysis buffer (2% SDS, 4% β-mercaptoethanol, 0.1% bromophenol blue, 10% glycerol, 1 M Tris-HCl (pH 6.8)). Samples were heated at 100°C for 10 min. Thereafter, 5 μL proteinase K (PK) (20 mg/mL) was added to the cooled samples, and incubated in a 55°C water bath overnight. The obtained LPS samples were run on 15% SDS-PAGE gels or 16% Tricine-SDS-PAGE gels and visualized by silver stain [52 (link)] and by Western blot, using mouse anti-Le^x (1:1500) and anti-Le^y (1:1500) antibodies (Santa Cruz). After incubation with a secondary rabbit anti-mouse peroxidase-conjugated IgM antibody (1:10,000) (Jackson ImmunoResearch), detection of the HRP conjugates was accomplished by chemiluminescence (Sigma) using a LAS-3000 Intelligent DarkBox (Fujifilm) (software Image reader LAS 3000 V2.2).

Proteins were resolved by SDS-PAGE on Criterion Precast gradient gels (4-15%, Biorad). and transferred to Hybond-C membrane (Amersham Biosciences). The membrane was incubated overnight at 4°C with either: (i) affinity purified anti-EMB-4, or anti alpha-tubulin (Accurate Chemical) antibodies diluted to 1:2000, in PBST-5% milk solution (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4, and 5% [w/v] dried milk). The membrane was incubated 1 h at room temperature with HRP-conjugated secondary antibodies (Jackson Immunoresearch) diluted to 1:5,000 in PBST and then visualized by Western Lightning ECL Kit from Perkin Elmer. Images were collected on a LAS-3000 Intelligent Dark-Box (Fujifilm).

BN (blue-native)-PAGE was performed using a Hoefer Mighty Small II SE 250 system (small gel: 10 cm×8 cm×0.15 cm) as described [17 (link),18 ]. The stacking gel had an acrylamide concentration of 3% and the separating gel an acrylamide gradient from 3.5 to 16%. Approximately 30 μg of solubilized purified CF₁F_O-ATP synthase in DDM was loaded per lane. A high molecular weight native marker (GE Healthcare) served as mass standard.
After electrophoresis the gel was scanned to document the bands stained by BN-PAGE. Subsequently, ATPase activity of the CF₁F_O-ATP synthase was determined by incubating the gel in buffer A1 containing 35 mM Tris, 270 mM glycine, 14 mM MgSO₄, 0.2% (w/v) Pb(NO₃)₂, 8 mM ATP, pH 7.8 at 37°C for several hours [19 (link),20 (link)]. The white lead phosphate precipitate was documented with a CCD-Imaging System (LAS 3000 intelligent dark box, Fujifilm).
SDS–PAGE was performed according to Laemmli [21 (link)], with a stacking gel of 3% and separating gel of 14%. In order to maintain integrity of the subunit c oligomer, protein samples were incubated at room temperature (25°C) in SDS loading buffer for 10 min. For visualization of protein bands, the gel was stained with Coomassie R-250.