Study Human Chemokine Receptor parallel with the other 96 samples. E) Long-term stabilization of proteins solubilized in 16 detergents for CCR5 is presented. A comprehensive list of detergents for Trx-hCRs is presented in Figure 5. The detergent effectiveness was carried out at 4uC using Trx-hCCR5 for 3 days, 1 week and 3 weeks, and detection was Dot blot using mouse anti-His6-tag monoclonal antibody. Dot blot intensities are shown as a 2-D column chart, and detergent numbers are from the Figure 5. F) Effectiveness of FC14 and FC12 on solubilization of Trx-hCCR3 (I) and hCX3CR1 (II) from cell lysate detected by Western blotting. Protein size standard is shown in the left lane. doi:10.1371/journal.pone.0004509.g006 ca.expasy.org) and lower than that of typical GPCRs with about 50% a-helical content [65,70]. It is possible that the contribution to the spectrum from the N-terminal fusion partner HP- thioredoxin may explain this difference. These results suggest that the purified Trx-hCCR3 folded with a reasonable secondary structure as calculated. Figure 7. Large-scale purification, identification and studying secondary structure of Trx-hCRs. A) Two-step purification of Trx-hCCR3 from membrane fractions. (I) Ni2+ affinity purification of His6-tagged proteins using a Hitrap chelating HP 5 ml column. (II) Gel filtration purification using Superdex 200 10/300 GL. Both purification steps were preformed on an A¨ KTA Purifier System. B) SDS-PAGE detection and Western blot identification of protein fractions of Trx-hCCR3. One of two duplicates of the SDS-PAGE was transferred onto a nitrocellulose membrane and subjected to Western blot using rabbit anti-human CCR3 monoclonal antibody. Lane Ni: 1/10 dilution of Trx-hCCR3 protein purified by Ni2+ affinity column (I); Lane A5 through A11: protein fractions of Trx-hCCR3 corresponding to gel filtration purification (II). All the samples were prepared by mixing 15 ml of protein fractions with 5 ml of SDS Sample buffer (46). 10 ml of BenchMark His-tagged protein standard was loaded as protein marker. SDS-PAGE was performed in a NuPAGE Novex 4–12% Bis-Tris Gel (in 16 MES buffer, Invitrogen) and stained by Simple Blue SafeStain (Invitrogen). C) Circular dichroism (CD) spectroscopy of purified Trx-hCCR3. Secondary structure of Trx-hCCR3 was analyzed by CD spectroscopy using purified Trx- hCCR3 monomer at 37uC with the concentration of 1 mg/ml in Buffer C. D) All 4 Trx-hCRs were purified and analyzed on SDS-PAGE with size markers and stained using Simple Blue SafeStain. E) Western blot identification of Ni2+ affinity column-purified Trx-hCCR5. Fraction of Trx-hCCR5 in the first step of purification was diluted to 0.1 mg/ml and subjected to Western blot detection using rabbit anti-human CCR5 monoclonal antibody. ECL DualVue Western blot Marker was used as protein standard. The 3 bands reveal that different states of Trx-hCCR5 were able to bind Ni2+ column. Judging from molecular weight, they may correspond to Trx-hCCR5 dimer, monomer, and truncated Trx-hCCR5, possibly, non-fused hCCR5. doi:10.1371/journal.pone.0004509.g007 PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4509