Figure 6. Detergent screening for solubilization of Trx-hCRs expressed by pBAD-hCRs in Top10.

A)–D) Effectiveness of the 96 detergents on solubilizing Trx-hCRs from cell lysates, as detected by Dot blot. Immunoblotting analysis was performed using mouse anti-His₆-tag monoclonal antibody. Each filter has 96 spots (a1 to h12), corresponding to the 96 detergents listed in Figure 5. Controls shown in (A) are positive control (+) using 1∶100 dilution of BenchMark His-tagged protein standard; negative control (−1) was pBAD-hCCR5 in Top10 cultured in the absence of inducer; negative control (−2) was L-arabinose induced sample solubilized without the addition of detergent. The negative controls were processed in 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 4°C 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.