and store at 220uC. These samples were used for detection of expression Dot blot. Stability of plasmids was also investigated for pEXP-hCRs in C41 and pBAD-hCRs in Top10. For each plasmid, 100 ml was taken from the cultures after 24 hours of expression, diluted in five ten-fold steps from each of which 100 ml was spread on LB-agar plates and LB-agar-ampicillin plates for overnight culture at 37uC. The following morning, plate pairs with about 100 colonies formed were used to calculate the stability ratio of the plasmid. Western blot & Dot blot detection. Sample preparation: stored cell pellets collected from 1 ml of culture were resuspended with 180 ml Lysis buffer. After three cycles of freeze-thaw at 280uC and 42uC, 20 ml of 10% detergent (FC14, unless otherwise stated) was added and mixed well, then, mixtures were incubated for 2 hours at 4uC with gentle shaking. After centrifugation at 16,000 g for 10 minutes the supernatant was removed into fresh tubes for protein expression testing. These samples were stored at 4uC if used within a few days, or at 280uC for future use. For Western blot detection, 15 ml of each sample was mixed with 5 ml of SDS sample buffer (46) and kept for 10 minutes at 37uC before applying to SDS-PAGE. Subsequently, the proteins in the gel were transferred onto nitrocellulose membrane following the standard protocol. For Dot blot detection, 3 ml of each of the samples was spotted onto nitrocellulose membranes and let dry at room temperature. All subsequent steps were the same for Western blot or for Dot blot. The nitrocellulose membrane was blocked in 20 ml of Block Buffer for 1 hour at room temperature, followed by washing twice for 5 minutes with 50 ml of PBST (16PBS, 0.1% Tween-20). For the primary antibody step, the membrane was incubated with 10 ml of 1:5000 diluted mouse His6-tag monoclonal antibody (or 1:2000 rabbit anti-human CCR3 or CCR5 monoclonal antibody) in Wash buffer for 1 hour, followed by 5 washes for 5 minutes with 50 ml Wash buffer. In the secondary antibody step, the membrane was soaked in 50 ml of 1:5000 diluted goat anti-mouse IgG/M HRP (or using 1:4000 goat anti-rabbit IgG HRP if the primary antibody was from rabbit) in Wash buffer for 50 minutes, followed by 5 washes for 5 minutes with 50 ml Wash buffer. Finally, the membrane was developed using ECL plus Western blot Detection Reagents (GE Healthcare) following the manufacturer’s protocol and imaged using a FluorChem Image System (Alpha Innotech Corp.). The signal intensities of Dot blot detection (spot intensity average) were quantified using the AlphaEaseFC software of the system. Detergent screening for solubilization of hCRs. Eighty- eight detergents were selected from the Solution Master Detergent Kit (Anatrace). Several detergents with too high CMC values were omitted from the screen. Additionally, HEGA-10, NP-40, Digitonin, and five kinds of detergent mixtures were included the list since they had proven effective in solubilization of other membrane proteins. In total, 96 detergents/detergent mixtures were listed and numbered as they appear in the Solution Master Detergent Kit with the eight additional ones appended as in Figure 5. All the detergents dissolved as 10% except those mixtures. For sample preparation of the four pBAD-hCRs expressed in Top10, 2 ml of overnight culture was inoculated in 200 ml of fresh TB medium and cultured at 37uC for about 3 hours with shaking, until the concentration reached an OD600 of 0.6,0.8. At this time, the temperature of the incubator was lowered to 16uC and the culture was continued for another 30 min, followed by the addition of 1 ml of 20% L-arabinose (to a final 0.05%) to induce the expression of pBAD-hCRs. After 24 hours of induction, the cells were harvested by centrifugation at 5000 g for 20 min. The cell pellets were resuspended completely with 20 ml of Lysis buffer and distributed into 96 1.5 ml of Eppendorf tubes with 180 ml/ Study Human Chemokine Receptor tube, numbered from 1 to 96. After 3 times of freeze-thaw at 280uC and 42uC, 20 ml of each detergent (final 1%) was added into the corresponding tube of cell lysate, except for those with higher CMC, where 40 ml of 2% detergent was added to 160 ml cell lysate. The samples were mixed carefully and incubated for 2 hours at 4uC with gentle shaking. Supernatants were collected after centrifugation at 16,000 g and 3 ml of each sample was applied to nitrocellulose membrane in the matrix form shown in Figure 6A from a1 to h12. Detection using Dot blot was performed as described above. Large-scale expression, solubilization and purification of synthetic hCRs. For high-level expression in 1 liter of culture, 10 ml of overnight culture was cooled on ice for 5 minutes and centrifuged at 3000 g for 15 minutes at 4uC. The supernatant was removed and the cell pellets were resuspend in 1 ml of fresh sterile LB medium, and was inoculated into 1 liter of fresh TB medium (containing 100 mg/ml ampicillin) for culture at 37uC with shaking at 220 rpm. When the cell concentration reached about 0.6 (OD600), the temperature was decreased to 16uC, and, the inducer was added after an additional 30 min shaking to start the expression of target proteins. The inducer concentrations were 0.3 mM IPTG for pEXP-hCRs in C41, or 0.05% L-arabinose for pBAD-hCRs in Top10. After 24 hours of expression, cells were harvested by centrifugation at 5000 g for 20 minutes, and stored at 220uC for future use. For collecting membrane fractions, stored cell pellets from 1 liter of culture were resuspended in 50 ml of Lysis Buffer followed by 3 times of freeze-thawing at 280uC and 42uC, and cells were lysed by passing through a French press at 18,000 psi. The cell lysate was centrifuged at 10,000 g for 1 hour to remove cell debris and inclusion bodies, then, ultra-centrifugation was applied for the supernatant at 100,000 g for 1 hour to collect the crude cell membrane fractions. These were resuspended in 20 ml Membrane Wash buffer, followed by another ultra-centrifugation at 100,000 g for 1 hour to collect the washed membrane fractions. Care was taken to resuspend the membrane pellets completely. The next step was solubilization, in which the washed membrane fractions were resuspended in 10 ml of Solubilization buffer containing one tablet of Protease Inhibitor Cocktail, followed by incubation overnight at 4uC with gently shaking. The following morning, the solution was again subjected to ultra-centrifugation at 100,000 g for 1 hour to remove non-solubilized proteins and collect the supernatant for purification. The supernatants were loaded on a Hitrap Chelating HP 5 ml column, which were charged with chelated Ni2+ and equilibrated with Buffer A for the first step of purification. After 15 column volumes (CVs) of washing with Buffer A plus 5% Buffer B (totally 50 mM imidazole), the target proteins were eluted with a linear gradient from 5% to 100% of Buffer B over 2 CVs. The fractions were tested by SDS-PAGE and those containing hCRs were pooled and concentrated using an Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-50 membrane (Millipore). To further improve the purity of the protein, the concentrated protein was loaded onto a Superdex-200 gel-filtration column equilibrated with Buffer C. The peak fractions from the elution were pooled and tested by SDS-PAGE or Western blotting. The concentration of purified proteins was measured with Nanodrop Spectropho- tometer (Thermo Scientific). Circular dichroism (CD) detection of secondary structure of Trx-hCCR3. CD experiments were preformed on Aviv 202 spectropolarimeter (Aviv Biomedical) with a 1 mm path length cell at 25uC. The purified protein sample came from gel filtration fractions and was concentrated to 1 mg/ml. The CD spectrum was recorded from 200 nm to 250 nm of wavelength with 1 nm PLoS ONE | www.plosone.org 13 February 2009 | Volume 4 | Issue 2 | e4509