Journal:

Article Title: The Alternative Sigma Factor ? B of Bacillus cereus : Response to Stress and Role in Heat Adaptation

doi: 10.1128/JB.186.2.316-325.2004

Figure Lengend Snippet: Overproduction and purification of σB. (Left panel) SDS-PAGE of protein extracts from E. coli BL21-Codonplus-(DE3)-RIL carrying either pET28-b or pMT01. (Right panel) Immunodetection of σB with anti-σB antiserum. Ten micrograms of protein was loaded for each sample. Lane 1, crude protein extract from E. coli carrying pET28-b; lane 2, crude protein extract from E. coli carrying pMT01; lane 3, crude protein extract from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 4, inclusion bodies isolated from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 5, representative fraction of purified σB after elution from an Ni2+ affinity column. The arrow indicates the position of the overproduced σB protein.

Article Snippet: Bio-Rad's broad-range prestained SDS-PAGE standards were used as molecular weight markers.

Techniques: Purification, SDS Page, Immunodetection, Isolation, Affinity Column

Journal:

Article Title: The Alternative Sigma Factor ? B of Bacillus cereus : Response to Stress and Role in Heat Adaptation

doi: 10.1128/JB.186.2.316-325.2004

Figure Lengend Snippet: Stress-induced activation of σB in B. cereus. (A) Cellular σB levels upon exposure to stress. Protein extracts from mid-logarithmic-phase B. cereus cells (lane 1) and stressed B. cereus cells were prepared as described in Materials and Methods. B. cereus cells in the mid-logarithmic growth phase were exposed to 4% (vol/vol) ethanol (lane 2), pH 5.2 (the pH was adjusted by addition of HCl) (lane 3), 2.5% (wt/vol) NaCl (lane 4), 50 μM H2O2 (lane 5), 1 mM diamide (lane 6), and 42°C for 30 min (lane 7). Proteins were also extracted from an overnight culture (lane 8). Forty micrograms of protein of each sample was loaded on the SDS-PAGE gel. Immunoblotting was performed with the sample by using the σB antiserum. (B) Relative amounts of σB. The signal intensities from the Western blot shown in panel A were quantified by using the Gel-Pro Analyzer software package (Media Cybernetics). The value for the mid-logarithmic-phase culture was defined as 1.

Article Snippet: Bio-Rad's broad-range prestained SDS-PAGE standards were used as molecular weight markers.

Techniques: Activation Assay, SDS Page, Western Blot, Software

Journal:

Article Title: The Alternative Sigma Factor ? B of Bacillus cereus : Response to Stress and Role in Heat Adaptation

doi: 10.1128/JB.186.2.316-325.2004

Figure Lengend Snippet: Effects of CCCP on growth, the intracellular concentration of ATP, and σB expression of B. cereus. (A) Growth (circles) and intracellular ATP concentration (bars) in B. cereus cells from a mid-logarithmic-phase culture (ML) after exposure to 1, 2, 10, and 50 μM CCCP (left panel) and after a heat shock (HS) at 42°C for 30 min (right panel). Growth of the culture was monitored by determining the increase in the OD600 during the 30 min of exposure to CCCP or 42°C. The increase in the OD600 over 30 min for an untreated culture was defined as 100%, and growth in the CCCP-treated and heat-shocked cultures was related to this value. Intracellular ATP concentrations were determined by the firefly luciferase assay as described in Materials and Methods. (B) Relative levels of σB in CCCP-treated B. cereus cells (lanes 1 to 5 contained 0, 1, 2, 10, and 50 μM CCCP, respectively) and heat-shocked B. cereus cells (lane 6). The cellular levels of σB were estimated by immunoblotting with anti-σB antiserum and quantification of the signal by the Gel-Pro Analyzer software package as described in the text. Forty micrograms of protein from each sample was loaded on the SDS-PAGE gel used for Western blotting.

Article Snippet: Bio-Rad's broad-range prestained SDS-PAGE standards were used as molecular weight markers.

Techniques: Concentration Assay, Expressing, Luciferase, Western Blot, Software, SDS Page