Induction, activity assay and determination of location of AP For the induction of AP, E. coli MPh42 cells were grown in the phosphate-less MOPS medium at 30°C, as described in [13]. At different instants of induction, an aliquot of 1.0 ml cell suspension was collected over 0.2 ml toluene and the activity of AP was assayed as described in [13], using GSK2126458 PNPP as the substrate. The amount of AP, which led to a change of absorbance of p-nitrophenol

by 0.1 per 6 min of enzyme-substrate reaction, had been considered as one unit of the enzyme [13]. For determination of the location of AP, the periplasmic, cytoplasmic and membrane fractions of cells were isolated from 1.0 ml of AP induced cell culture, as described in [20]. After electrophoresis of the fractions in 12% SDS-polyacrylamide

gel, ‘western blot’ experiment with anti-AP antibody was performed. Isolation of aggregated proteins Isolation of total soluble (containing dispersed protein pool) and insoluble (containing aggregated protein pool) cell fractions was based on the method described in [21]. Cells were allowed to grow at 30°C in MOPS medium up to bacterial OD600 nm ~0.5. 25.0 ml of grown culture was rapidly cooled to 0°C and centrifuged at 4°C for 10 min at 6000 rpm. The cell pellet was re-suspended SRT1720 molecular weight in 80 μl of buffer A [10 mM potassium phosphate buffer (pH-6.5); 1.0 mM EDTA; 20% (w/v) sucrose and 1.0 mg/ml lysozyme] and incubated for 30 min on ice. To the cell suspension, 720 μl of buffer B [10 mM potassium phosphate buffer (pH-6.5); 1 mM EDTA] was added and the cells were dipped in ice to sonicate by microtip ultrasonicator (using level 2, 1 min, 50% duty, three cycles). Intact cells were removed by centrifugation at 2000 g for 15 min at 4°C. The supernatant was YM155 purchase further centrifuged at 15000 g for 20 min at 4°C and the pellet was collected. The pellet, which contained membrane and aggregated proteins, was washed with and finally re-suspended by brief sonication in 320 μl of buffer B. 80 μl of 10% (v/v) NP40 was then added to the suspension, mixed well and centrifuged

at 15000 g for 30 min at much 4°C to isolate the aggregated proteins as the pellet and to remove the membrane proteins as supernatant. The steps of re-suspension in buffer B, addition of NP40 and subsequent centrifugation were repeated three times. NP40-insoluble aggregated protein pellets were washed with 400 μl buffer B and finally re-suspended in 200 μl of buffer B. Isolation and purification of sigma-32 The isolation and purification of the His-tagged sigma-32 from E. coli strain BB2012, using the Ni2+-NTA agarose column, were carried out according to [22]. Immunization The antibodies of AP and sigma-32 were raised separately according to the method of Oliver and Beckwith [19] as described in [13].