Quaternary Structure and Lipid Affinity of PLC Figure 7. Secondary structure analysis of CrPLC. (A) Comparision of Far UV-CD spectra recorded for dimer and monomer fractions of CrPLC WT, CrPLC C7A mutant and equimolar fractions of mixed dimer and monomer CrPLC represented in different line patterns as indicated in inset. (B) Bar graph represent the calculated percentage secondary structure content including a-helix, b-sheets, turns and random coil content in CrPLC WT (monomer, dimer and dimer : monomer 1:1) and CrPLC C7A mutant. doi:10.1371/journal.pone.0039258.g007 with 0.3 mM IPTG at cell culture O.D600 of 0.6. Protein expression was carried out at 16uC for 48 hrs. Recombinant proteins were purified by immobilized metal affinity chromatog- raphy using Co2+ metal ion resins (Clontech, Laboratories Inc. USA) according to manufacturer’s protocol. Chlamydomonas Culture and Cell Homogenate Preparation C. reinhardtii strain cc 124 mt (2) used in this study was obtained from Chlamydomonas resource center (Department of Plant Biology, University of Minnesota). Culture was grown on TAP (tris-acetate phosphate media, pH 7.4 and supplemented with Hutners trace elements) at 25uC in an incubator shaker (120 rpm) with continuous exposure of fluorescent white light (2300 Lux). Cells in early exponential growth phase were used for all the experiments. Chlamydomonas total cell lysate (Cr TCL) was prepared by suspending cells in phosphate buffer saline (PBS; 150 mM NaCl, 10 mM sodium phosphate, and pH 7.2) in presence of protease inhibitor cocktail. Cell lysis was performed by sonication, giving 8 sec on and 8 sec off pulse for 6–8 times. Samples were solubilized by incubating crude lysate with 263% SDS Laemmli buffer for 30 min at 60uC. Size-exclusion Chromatography Size exclusion chromatography was performed with a Akta explorer chromatography system (GE Healthcare USA) on HiLoad 16/60 Superdex 200 (1.0630 cm) prep grade column and superose 12 10/300 GL analytical grade column, using FPLC mode, equilibrated with PBS, operated with flow rate of 1 ml/min at 4uC. Monomer and dimer species were determined by comparing it with standard molecular weight marker (Bio-Rad, USA), separated under the isocratic conditions. The peaks in the chromatogram were fit to a Gaussian curve with the IGOR Pro program (Wave Metrics Inc.’s). Western Blotting Protein blotted nitrocellulose membrane was blocked using 5% non-fat dry milk powder in PBS supplemented with 0.1% tween- 20 for 1 hr at room temperature. Blocked nitrocellulose mem- brane was then incubated with primary antibody generated against recombinant CrPLC at a 1:3000 dilution. Immunolabel- ling was detected by using HRP (horseradish peroxidase) conjugated anti-rabbit secondary antibody (1:5000 dilution) and visualized by enhanced chemiluminiscence method using the standard protocol. PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39258