Bas2500 bio imaging analyzer

RNA was 5′-end labeled using T4 polynucleotide kinase and [γ-³²P]ATP and then it was purified by denaturing polyacrylamide gel electrophoresis (PAGE). The RNA concentration was determined by the UV absorbance at 260 nm. RNA was annealed by heating at 90 °C and slow cooling to room temperature in sterile water. RNA, helicates, and the ADP-1 peptide were mixed in a buffer (5 μL) containing Tris-HCl (50 mM, pH 8.0), KCl (50 mM), DTT (100 mM), and Triton X-100 (0.05%) and incubated for 10 min on ice. The samples were analysed by electrophoresis on 15% polyacrylamide (PAA) gels in 0.5 × TB buffer and electrophoresed at 5 W and 5 °C. Gels were exposed to a phosphor imaging plate and scanned with a FUJIFILM BAS-2500 bio-imaging analyzer.

DNA footprinting was performed using purified F-HA-Zic2 proteins^{22 (link)} and DNA fragments with the Tgif1 5′ region, which was digested from the pBS-Tgif plasmid. The fragment was digested at the BamHI and HindIII sites, and was labelled with ³²P with T4 polynucleotide kinase. After the reaction, the BamHI side was digested at the EcoRI inner site and separated by native gel electrophoresis^{54 (link)}. The ³²P labelled 1 nM DNA fragments were incubated in the presence or absence of 12.5 nmol F-HA-Zic2 protein in the reaction buffer (20 mM Tris-HCl pH8.0, 3 mM MgCl₂, 5 mM CaCl₂, 100 mM NaCl, 100 mM DTT, 0.1 mM EDTA, and 50 μg/ml bovine serum albumin) for 30 min at 25 °C. DNase I (20 ng) was added and incubate for 60 s at 25 °C. The samples were extracted with phenol/chloroform/isoamyl alcohol, and precipitated with ethanol. DNA was analysed by 8 M urea 8% polyacrylamide gel electrophoresis together with marker DNA, which was digested at GTP by the Maxam-Gilbert method^{54 (link)}. The gel was analysed with the Bioimaging Analyzer BAS2500 (Fuji).

The in vitro lipase activity was measured as previously described with some modifications [30 (link)]. To examine phospholipase activity of AtDSEL, the recombinant fusion protein was incubated with 1-palmitoyl-2-¹⁴C-palmitoyl-PC (2.22 GBq/mmol; GE Healthcare, Uppsala, Sweden) in 50 mM sodium phosphate buffer (pH 5.8). Lipids were extracted and separated by thin layer chromatography (TLC) (Silica Gel 60; Merck). Radioactive products were detected using the Bio-Imaging Analyzer (BAS2500; Fuji Photo Film). To determine substrate specificity, the MBP-AtDSEL was incubated with 250 μM of various lipid substrates having sn-1 or sn-2 acyl chains, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), trilinolein, triolein, 1,2-diacylglycerol (1,2-DAG), 1,3-diacylglycerol (1,3-DAG), 1-monoacylglycerol (1-MAG), 2-monoacylglycerol (2-MAG), and triacylglycerol (TAG) in 200 μL of 50 mM sodium phosphate buffer (pH 7.2) for 30 min at 30 °C. The released free fatty acids were extracted and reacted using the nonesterified fatty acid (NEFA) colorimetric kit (Wako Pure Chemicals) and measured colorimetrically at 546 nm.