For western blotting, cells were chilled on ice, rinsed with ice-cold PBS twice and lysed with RIPA buffer (Sigma-Aldrich, R0278) plus the protease inhibitor cocktail. Protein concentrations were determined using the BCA Protein Assay Kit (BioRad). About 25 μg protein per sample was loaded on a 10% SDS-PAGE gel and transferred to the PVDF membrane, which was washed with PBS Tween 20 buffer (PBST) (Thermo Fisher Scientific, #28360) for 5 minutes three times, blocked with 5% milk in PBST, and incubated with the anti-HBsAg (Novus, #NB100-62652) or the antibody directed against denatured core protein in the cold room overnight. The anti-core antibody was prepared in our lab using the recombinant core protein (Ou et al., 1989 (link)). After washing with PBST 3 times for 10 minutes each, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Abcam) at room temperature for one hour. The membranes were washed with PBST three more times with 10 minutes per wash and subjected to chemiluminescent western blot analysis. All experiments were repeated at least three times.
Anti hbsag
Manufactured by Novus Biologicals
Sourced in United States
Anti-HBsAg is a laboratory reagent used to detect the presence of hepatitis B surface antigen (HBsAg) in biological samples. It is a key component in serological testing for hepatitis B virus infection.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Abcam (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Bca protein assay kit, by Bio-Rad (2 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Millicell ez slide, by Merck Group (1 mentions)
All in one fluorescence microscope, by Keyence (1 mentions)
Anti ck19, by Proteintech (1 mentions)
Anti ck7, by Abcam (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Anti hbx, by Abcam (1 mentions)
Anti alb, by Proteintech (1 mentions)
4 protocols using anti hbsag
1
Western Blotting Procedure for HBsAg and Core Protein
2
Immunofluorescence Analysis of Mitochondrial and Viral Proteins
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Cell fixation was performed using 4% Paraformaldehyde Phosphate Buffer Solution for 10 min at room temperature (RT). Blocking was performed with 2.5% normal horse serum (Vector Laboratories, Inc., Burlingame, CA, USA) for 1-h at RT. Immunofluorescence analysis of MT-CO3, hepatitis B surface protein (HBs), and HBc was performed using 4-well Millicell® EZ slides (Merck Millipore, Ltd., Carrigtwohill, Ireland) with one of the following primary antibodies for overnight at 4°C: rabbit polyclonal anti-MT-CO3 (1:200; Cat#55082-1-AP; Proteintech, Rosemont, IL, USA), rabbit polyclonal anti-HBsAg (1:200; Cat#NB100-62652; NOVUS Biologicals, Littleton, CO, USA), and mouse anti-HBcAg (1:100; Cat#ab8638; Abcam, Cambridge, MA, USA). As secondary antibodies, Vecta Fluor Excel Amplified Anti-Rabbit IgG, DyLight 488 Antibody Kit(Vector Laboratories, Inc.), and Vecta Fluor Excel Amplified Anti-Mouse IgG, DyLight 488 Antibody Kit(Vector Laboratories, Inc.) were used. Incubated at room temperature for 15 min with Amplifier Antibody, followed by 30 min at room temperature with VectaFluor Reagent. Cells were observed using an all-in-one Fluorescence Microscope (KEYENCE Corp., Osaka, Japan).
3
Western Blot for Liver Markers
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An equal amount of total protein was run on 10% SDS-PAGE (EpiZyme, cat. no. PG112), transferred to PVDF membranes (Merck millipore, cat. no. IPVH00010) (380 mA for 2 h), and probed with primary antibodies. The primary antibodies include anti-HBsAg (1:1000, 27 kDa, Novus Biologicals), anti-HBx (1:1000, 17 kDa, Abcam), anti-CK7 (1:1000, 51 kDa, Signalway antibody (SAB)), anti-CK19 (1:1000, 40 kDa, Signalway antibody (SAB)), anti-Hep-Par1 (1:1000, 165 kDa, Proteintech Group), anti-ALB (1:1000, 66 kDa, Proteintech Group), anti-α-Tubulin (1:1000, 55 kDa, Proteintech Group), anti-GAPDH (1:1000, 36 kDa, CST). The target protein bands were captured by binding of the secondary antibodies linked with peroxidase (1:1000, Anti-Rabbit IgG (H + L), CST) (1:1000, anti-mouse IgG (H + L), CST) to the primary antibodies.
4
Western Blotting Procedure for HBsAg and Core Protein
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For western blotting, cells were chilled on ice, rinsed with ice-cold PBS twice and lysed with RIPA buffer (Sigma-Aldrich, R0278) plus the protease inhibitor cocktail. Protein concentrations were determined using the BCA Protein Assay Kit (BioRad). About 25 μg protein per sample was loaded on a 10% SDS-PAGE gel and transferred to the PVDF membrane, which was washed with PBS Tween 20 buffer (PBST) (Thermo Fisher Scientific, #28360) for 5 minutes three times, blocked with 5% milk in PBST, and incubated with the anti-HBsAg (Novus, #NB100-62652) or the antibody directed against denatured core protein in the cold room overnight. The anti-core antibody was prepared in our lab using the recombinant core protein (Ou et al., 1989 (link)). After washing with PBST 3 times for 10 minutes each, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Abcam) at room temperature for one hour. The membranes were washed with PBST three more times with 10 minutes per wash and subjected to chemiluminescent western blot analysis. All experiments were repeated at least three times.
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