Hpv16 l1 protein

Equal amounts (500 ng) of HPV16 L1 protein (Abcam), purified L1:P18I10 and L1:T20 VLPs were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and then transferred to a PVDF membrane (Millipore, Burlington, MA, USA) using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000, anti-HIV-1 gp120 V3 loop mAb (NIBSC, EVA3012) at a dilution of 1:40 and HIV1 gp41 (2F5) mAb (NIBSC, ARP3063) at a dilution of 1:4000, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The Western ECL substrate kit (BIO-RAD) was used for signal development. The blot images were acquired by using Odyssey Fc imaging system at a chemiluminescence channel.

Equal amounts (200 ng) of HPV16 L1 protein (Abcam) and L1:P18I10 VLPs purified from both methods were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and were then transferred to a PVDF membrane (Millipore) using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000 and anti-HIV1-V3 loop mAb (NIBSC, EVA3013) at a dilution of 1:500, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad). The blot images were acquired by using Odyssey Fc imaging system.