Anti xpress antibody

The Ni-coated plate (HIS-Select High Capacitty (HC) Nickel coated plates; Sigma-Aldrich) was blocked with Super Block (Pierce), then dried and kept at 4°C until use. The PEG-precipitated culture supernatants or the cell lysates were loaded onto the 96-well Nickel-coated plate and incubated at 37°C for 1 h followed by washing three times with TBS-T (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20; Tris-buffered saline containing 0.1% Tween 20). The plate was then incubated for 30 min at 37°C with a mouse mAb (anti-Xpress^™ antibody; Invitrogen) and again washed three times with TBS-T. The plate was reacted with anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Dako) for 30 min at 37°C and washed with TBS-T three times. Finally, the color reaction was developed with a substrate solution (100 μl 0.0015% H₂O₂ plus 120 μg/ml TMB [3,3',5,5'-tetramethylbenzidine]). The optical density (OD) was measured at 450 nm with subtracted the reference wavelength (OD₆₃₀) by using a Spectra Max 190 microplate spectrophotometer (Molecular Devices) after stopping the reaction with 50 μl 3N H₂SO₄.

The GT335 (1:500 dilution) and polyE (1:200 dilution) antibodies were obtained from AdipoGen (San Diego, CA); anti-anti-acetylated α-tubulin (1:1000 dilution) and anti-polyglutamylated tubulin B3 (1:500 dilution) were obtained from Sigma-Aldrich; anti-detyrosinated antibody was obtained from Abcam; anti-Xpress antibody (1:200 dilution) was procured from Invitrogen. Anti-RPGR antibody (1:500 dilution) was raised against an N-terminal epitope, which is common to both RPGR^const and RPGR^ORF15 isoforms. Detailed characterization of this antibody was previously reported (Ghosh et al., 2010 (link); Rao et al., 2015 (link)).

Reactivity of HBsAg from the transfected cell lysates and the culture supernatants was determined by using commercial ELISA kits (a Rapid II kit from Beacle Inc., Kyoto, and HBs ELISA Kit from Bioneovan Co., Ltd., Beijing) according to the manufacturer’s instructions. In Western blotting analysis, the respective protein samples prepared from the transfected cell lysates and the culture supernatants were mixed with 5X sample buffer (the final concentration was 1X) and boiled for 5 min at 100°C. The protein separated by SDS-polyacrylamide gel (4–12%) electrophoresis was then transferred to a PDVF membrane (Cat #162–0177; Bio-Rad) and blocked with 5% skim milk in TBS-T. The expressed HBsAg was detected by a mouse mAb (anti-Xpress^™ antibody; Invitrogen) as a primary antibody followed by HRP-labeled goat polyclonal anti-mouse immunoglobulins (Dako) as a secondary antibody. Both antibodies were diluted at 1:5000 in solution 1 and solution 2 (Can Get Signal^™; Toyobo), respectively. Chemiluminescence was visualized by Chemi-Doc (Bio-Rad).

Recombinant pkMSP-3 were resolved by 12% SDS-PAGE and stained with Coomassie brilliant blue (Bio-Rad, USA). The separated proteins were also electrophorectically transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA) with protein amounts ranging between 50 to 300 ng and blocked overnight in Tris Buffered Saline (TBS) containing 5% skimmed milk at 4°C. The membranes were washed three times with TBS containing 0.2% Tween-20 (TBS-T) and probed with either anti-Xpress™ antibody (1:5000 dilution) (Invitrogen, USA) or with patient serum (1:200 dilution) with TBS containing 2.5% skimmed milk for two hours with constant shaking at room temperature. After three washes with TBS-T the membrane was treated with biotin-labelled goat anti-mouse IgG (1:2500 dilution) if using anti-Xpress™ antibody; or biotin labelled goat anti-human IgA plus IgM plus IgG (1:2500 dilution) is using human serum (Kierkegaard and Perry Inc., USA) for one hour, followed by streptavidin-alkaline phosphatase (1: 2500 dilution) for one hour. Finally, the membranes were developed by chromogenic nitro-blue tetrazolium/5-bromo-4-chloro-3’-indolyphosphate substrate. The colour was allowed to develop at room temperature in the dark.