Cell debris was removed by centrifugation and then the https://www.selleckchem.com/products/cftrinh-172.html sample was washed and concentrated (to half of the total volume) by using a Microcon® YM-3 filter unit (10,000 × g, 4°C). selleck inhibitor Protein quantitation was performed using Bio-Rad Protein Assay® system. A total of 500 μg of proteins was precipitated through Ready-Prep 2D Cleanup Bio-Rad® kit. Precipitated proteins were resuspended in 300 μL IEF buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.0002% bromophenol blue) followed by the addition of DTT to 100 mM and 0.2% Bio-Rad ampholytes and the sample mix was incubated for 1 h at 25°C. The entire volume was loaded in the Protean® IEF focusing tray (17 cm) using the following

strips pH ranges: 4.7-5.9/5-8/3-10NL (ReadyStrip™ IPG) that were actively rehydrated at 50 V for 12 h. The focusing step was performed at 250 V for 15 min; 2,000 V for 2 h; 8,000 V for 4 h and finally 10,000 V for 11 h, all the steps at 20°C. Focused proteins in the strip were then incubated at 25°C with gentle agitation for 15 min in equilibrium buffer (6 M urea; 2% SDS; 0,05 M Tris/Cl pH 8.8; 20% glycerol) containing 2% DTT and then 15 min in equilibrium buffer containing 2.5% iodoacetamide. Finally, the strip was placed onto a 12.5% polyacrilamide gel for the second dimension in Protean® II (Bio-Rad) system at 50 V for 23 h. The gels were fixed for 1 h (50% ethanol; 2% phosphoric acid), stained for 3 h (0,12% CBB G-250; 10% phosphoric acid; 10% ammonium sulphate; 20% methanol) and then washed

three times with 15% methanol. Digital images of the gels were analyzed and spots quantified using Delta2D v.3.6 software. Spot volume www.selleckchem.com/products/sbi-0206965.html was normalized as a percentage of the total volume of all spots on the corresponding gel and also manually confirmed. The threshold for accepting a meaningful variation was a factor of 2.0 (p < 0,05). A total of 81 proteins spots showing differences in the expression pattern between control and polyP(-) strains (three independent replicates) were selected for further MS analysis. In-gel protein digestion and sample preparation

Spots of interest from Coomassie blue-stained 2D gels were excised manually, deposited in 96-well plates and processed automatically in a Proteineer DP (Bruker Daltonics, Bremen, Germany). The digestion 17-DMAG (Alvespimycin) HCl protocol used was based on Schevchenko et al. [48] with minor variations: gel plugs were washed firstly with 50 mM ammonium bicarbonate and secondly with acetonitrile (ACN) prior to reduction with 10 mM DTT in 25 mM ammonium bicarbonate solution, and alkylation was carried out with 55 mM IAA in 50 mM ammonium bicarbonate solution. Gel pieces were then rinsed with 50 mM ammonium bicarbonate and with ACN, and then dried under a stream of nitrogen. Modified porcine trypsin (sequencing grade; Promega, Madison WI) was added at a final concentration of 16 ng/μl in 25% ACN/50 mM ammonium bicarbonate solution and the digestion took place at 37°C for 6 h. The reaction was stopped by adding 0.5% TFA for peptide extraction.