1d image analysis software

The protein concentration was determined using the method established by Bradford [20 (link)]. Determination of the protein concentration was performed in triplicate and the absorbance was measured at 595 nm. The protein concentration (mg/μL) was determined from linear regression calculations based on the values obtained from the standard curve. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed using 14% (w/v) gels. Electrophoresis was carried out at 20 mA/gel in Tris-glycine buffer (pH 8.3) containing 0.01% SDS. The molecular mass standard proteins used included phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). Gels were stained with Coomassie blue R-250, 0.2% (w/v). The relative molecular mass of BmooPAi was estimated using Kodak 1D image analysis software.

The DNA-nicking method was described previously25 (link) with some modifications. Briefly, a typical DNA-nicking reaction mixture (320 μL) contained 20 mM Tris-acetate (pH 7.9 at 25 °C), 10 mM magnesium acetate, 1 mM DTT, a negatively supercoiled DNA template, and LacI. Where specified, 1 mM of IPTG was also added to the DNA-nicking assays. All components were assembled on ice and incubated for 30 min at 37 °C. After the incubation, the supercoiled DNA templates were digested by either Nt.BbvCI or Nb.BbvCI at 37 °C for 30 min or various times. Then, a large excess of a double-stranded oligonucleotide containing Nt.BbvCI recognition site were added to the reaction mixtures to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by extraction with an equal volume of phenol. The DNA samples were precipitated with ethanol and dissolved in 25 μL of 10 mM Tris-HCl buffer (pH 8.5). The linking number of the ligated DNA products was determined with 1% agarose gel electrophoresis in the absence or presence of 0.5 μg/ml of chloroquine and calculated from the gel images stained with SYBR Gold using KODAK 1D Image Analysis Software.