The resulting peptides were extracted from Sirolimus mw the gel plug with 0.1% (v/v) trifluoroacetic acid/50% (v/v) acetonitrile. Digests were spotted on a MALDI target using α-cyano-4-hydroxycinnamic acid as a matrix. Spectra were acquired on a 4800 MALDI TOF/TOF analyser (Applied Biosystems, Foster City, CA). Data
analysis and MS database searching were performed using GPS Explorer™ and mascot software. Total RNA was isolated from P. gingivalis cells grown to mid-exponential phase (OD600 nm of c. 1.0) by using an RNeasy Mini kit (Qiagen Science). DNA was removed with RNase-Free DNase. cDNA was generated in a reaction mixture containing a random primer (Promega), dNTP mixture, RNase inhibitor, DTT, Superscript III Reverse Transcriptase (Invitrogen) and
DEPC-treated water. Real-time qPCR was performed using Brilliant SYBR Green II QPCR Master Mix (Stratagene) with an Mx3005P™ Real-Time PCR System (Stratagene) according to the manufacturer’s instructions. Primers for the real-time qPCR are listed in Table S1 and were designed using the primer3 program. Real-time qPCR conditions were as follows: one cycle at 95 °C for 10 min, and 35 cycles of 95 °C for 30 s and 60 °C for 1 min. At each cycle, accumulation of PCR products was detected by the reporter dye from the dsDNA-binding SYBR Green. To confirm that a single PCR product was amplified, after the PCR, a dissociation curve (melting curve) was constructed in the range 55–95 °C. All data were analysed using Mx3005P software. The expression level of each targeted AG-14699 gene was normalized to that of the 16S rRNA gene, which was used as a reference. All
PCR reactions were carried out in triplicate. The efficiency of primer binding was determined by linear regression by plotting the cycle threshold (CT) value versus the log of the cDNA dilution. Relative quantification of transcript was determined Phosphoglycerate kinase using the comparative CT method () calibrated to 16S rRNA gene. qPCR experiments were performed multiple times independently, yielding comparable results. We constructed an rgpA rgpB kgp porK mutant from an rgpA rgpB kgp strain and compared secreted proteins between the rgpA rgpB kgp and rgpA rgpB kgp porK strains to avoid degradation of secreted and surface proteins by gingipains as the wild-type strain secreted gingipains that had the ability to process both secreted and surface proteins, while the porK mutant secreted no gingipains. 2D-PAGE of the particle-free (membrane-free) culture supernatants from the kgp rgpA rgpB and kgp rgpA rgpB porK mutants was performed. As a control, three protein spots in each 2D gel, which exhibited the same amounts of proteins with the same molecular masses and isoelectric points, were subjected to MALDI-TOF mass analysis, resulting in the same proteins (PGN_0916, PGN_1367 and PGN_1587; Fig. 1). Their molecular masses and isoelectric points calculated from their amino acid sequences were 69 044 and 4.88 for PGN_0916, 49 199 and 5.