Western blotting was performed following a standard protocol described previously.18 (link) Briefly, total proteins were extracted from cells by RIPA lysis buffer (Abcam, Cambridge, UK) and protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Abcam). Equal amounts of protein (25 µg) resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk for 1 hour at room temperature and then incubated with primary antibodies for overnight at 4°C. The next day, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Antibodies used in this study included anti-TBK1 (108A429; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-β-actin (C-4; Sigma), anti-phospho-TBK1 (D52C2; Cell Signaling, Danvers, MA, USA), anti-ITK (D3H5; Cell Signaling), suppressor of cytokine signaling 1 (anti-SOCS1, A156; Cell Signaling), anti-phospho-SOCS1 (Cell Signaling), anti-IRF-3 (D83B9; Cell Signaling), anti-HBsAg (ab20402; Abcam). Anti-β-actin was used as an internal control.
Anti hbsag
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-HBsAg is a laboratory product used for the detection and quantification of hepatitis B surface antigen (HBsAg) in biological samples. It is a key component in the diagnosis and monitoring of hepatitis B virus (HBV) infection.
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Lab products found in correlation
Anti hbcag, by Abcam (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Anti phospho tbk1 d52c2, by Cell Signaling Technology (1 mentions)
Anti irf3 d83b9, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Abcam (1 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti irf3, by Cell Signaling Technology (1 mentions)
Manganese 2 chloride tetrahydrate mncl2 4h2o, by Merck Group (1 mentions)
Apccy7 conjugated anti cd8, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Anti tbk1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Parp1, by Abcam (1 mentions)
Cd8 primary antibody, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
10 protocols using anti hbsag
1
Western Blotting Procedure for Protein Analysis
2
Immunofluorescence Assay for HDAg and HBsAg
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Phase contrast pictures were taken using the Cytation™ 5. For immunofluorescence assays, cells were cultured on collagen-coated glass coverslips and fixed for 10 min in 4% paraformaldehyde. Permeabilization with PBS complemented with 0.2% TritonX-100 for 30 min was followed by incubation with blocking solution (PBS supplemented with 3% BSA and 10% FBS) for 30 min. HDAg was detected by incubation with human anti-HDAg serum for 1 h at RT, followed by incubation of 1 h at RT with Alexa Fluor™ 568 Goat anti-Human IgG diluted at 1:1000 (Thermo-Fisher, St-Laurent, QC, Canada). HBsAg was detected by incubation of cells with anti-HBsAg diluted at 1:150 (Abcam, Toronto, ON, Canada #ab9193) followed by incubation with Alexa Fluor® 488 AffiniPure Goat Anti-Horse IgG diluted at 1:10,009 (Thermo-Fisher, St-Laurent, QC, Canada). Cell nuclei were stained with DAPI. Coverslips were mounted on microscope slides using Prolong antifade reagent (Thermo Fisher, St-Laurent, QC, Canada). Cells were analyzed using a confocal microscope (Zeiss LSM 780, Dorval, QC, Canada).
3
Western Blot Analysis of Recombinant Protein Expression
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To demonstrate that the recombinant plasmid was really expressed in muscle cells of injected fish, we perform a western blot with anti-HBSAg antibody (10-1323, Fitzgerald Industries International, North Acton, MA, USA).The mixed muscle tissues from three fish of each group were used for Western blot analysis. Protein was extracted from muscle tissue and the protein concentration was evaluated using a BCA protein assay. Equal amounts of protein were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Immunoblotting was carried out using a 1:1000 dilution of antibodies anti-HBSAg (Abcam, Cambridge, UK) and anti-β-actin (Abcam) according to the manufacturer’s instructions. Primary antibodies were visualized using the enhanced chemiluminescent development reagent (Amersham Pharmacia Biotech Ltd., Little Chalfont, UK) following the peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
4
Antibody and Reagent Sources for HBsAg Study
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Anti-TBK1 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β actin was obtained from Sigma (St. Louis, MO, USA). Anti-Phospho-TBK1 (pTBK1, Ser172), anti-phospho-IRF-3 (Ser396), and anti-IRF-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-HBsAg was purchased from Abcam (Shanghai, China). For flow cytometry, PE-conjugated anti-IFN-γ and APC-CY7-conjugated anti-CD8 were purchased from eBioscience (San Diego, CA, USA). Manganese (II) chloride tetrahydrate MnCl2·4H2O and manganese (II) acetate dehydrate Mn (OAc)2·2H2O were obtained from Sigma-Aldrich (St. Louis, MO, USA). The HBsAg was purified from CHO cells expressing recombinant HBsAg vaccine (rHBVvac; China North Pharmaceutical Group Corporation, Shijiazhuang, China) as described previously [13 (link)].
5
Comprehensive Antibody Procurement Protocol
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Ku70, Ku80, PARP1, IRF1, cGAS, STING primary antibodies, anti-HBsAg, and anti-HBcAg were purchased from Abcam. IRF3, IRF7, RNA pol III, CCL3, and CCL5 antibodies were purchased from Santa Cruz. Secondary antibodies labeled with Alexa Flour were all purchased from Invitrogen. Mouse antibodies were all purchased from BD Bioscience, except for CD8 primary antibody from eBioscience. Refer to Table S1 in Supplementary Material for detailed information.
6
Isolation and Quantification of HBV Virions
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Harvested supernatants from transfected HepG2 cells were added onto 30% sucrose solution and concentrated by ultracentrifugation at 200,000 ×g for 15 h at 4 °C. The viral particles were resuspended with appropriate volume of TNE buffer. The viral suspensions were precleared by addition of Dynabeads Protein G (Invitrogen). After the removal of beads, anti-HBsAg (Abcam) and anti-preS2 antibodies (Abcam) were added at a ratio of 1:2 and incubated overnight at 4 °C. Dynabeads Protein G were added again and incubated at 4 °C for 4 h. The beads were washed with PBS for 8 times, resuspended in core lysis buffer and digested by proteinase K (20 mg/mL) at 50 °C for 1 h. Finally, HBV virion DNA was extracted by phenol-chloroform and determined by qPCR as described above.
7
Western Blot Analysis of Cellular Proteins
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A NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Rockford, IL) was used to extract nuclear and cytoplasmic proteins according to the manufacturer’s protocol. Each 20 μg protein sample was separated on an 8/10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Darmstadt, Germany) using the Trans-Blot Turbo™ blotting system (Bio-Rad). After transferring, membranes were blocked with 5% BSA at room temperature for 2–3 h. The membranes were then incubated overnight at 4°C with anti-SREBP-2 (1:600; Abcam, Cambridge, UK), anti-HMGCR (1:5,000; Abcam), anti-LDLR (1:500; Abcam), anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) (1:600; Proteintech, Wuhan, China), anti- CYP-7α (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HBsAg (1:1,000; Abcam), anti-HBV xAg (1:200; Santa Cruz Biotechnology), anti-Hep B cAg (1:200; Santa Cruz Biotechnology), anti-GAPDH (1:10,000; Abcam), or anti-lamin B (1:300; Boster, Wuhan, China) primary antibodies, respectively. After washing with TBST, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (1:5,000; Boster). An enhanced chemiluminescence Western blotting substrate kit (BioVision, SanFrancisco, CA) was used to detect specific proteins.
8
Immunofluorescence Labeling of Tetracycline-Induced L. tarentolae Cells
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For immunofluorescence labeling, tetracycline-induced L. tarentolae cells were washed with PBS and fixed in 4 % paraformaldehyde for 30 min at RT. Lysine-coated glass coverslips were covered with fixed cell suspension and left to dry at RT. Next, the cells were permeabilized with 0.2 % Triton X-100 in PBS for 10 min at RT. Subsequently, the coverslips were incubated with primary rabbit anti-HBsAg (Abcam) and mouse anti-E2 AP33 (kindly provided by A. Patel) antibodies diluted to 1:1000 in PBS-BSA buffer [0.5 % (w/v) BSA] for 1 h at RT. The coverslips were then washed with PBS and incubated with Alexa Fluor 633-labeled goat anti-mouse and Alexa Fluor 488-labeled goat anti-rabbit secondary antibodies (1:1000 in PBS-BSA) for 1 h at RT. After washing, the coverslips were mounted onto microscope slides with ProLong Gold antifade reagent.
9
Immunohistochemical Analysis of HBsAg and AFP in Liver Tissue
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For the HBsAg and AFP staining, liver tissue sections of formalin-fixed and paraffin-embedded were stained with an anti-HBsAg (Abcam, Cambridge, UK) primary antibody or anti-AFP (ProteinTech, America) overnight at 4 ℃. Then sections were washed and stained with a biotinylated secondary immunoglobulin G antibody and a streptavidin–horseradish peroxidase conjugate (ZSGB-BIO) in accordance with the manufacturer’s instructions. Sections were developed by a diaminobenzidine peroxidase substrate kit (Vector, Burlingame, CA).
10
Immunofluorescence Imaging of Hepatitis B Viral Proteins
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HepG2.2.15 cells plated on coverslips were washed with PBS and fixed with 4% (v/v) formaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature followed by blocking in TBS-T solution (25 mM Tris, pH 7.4, 3.0 mM KCl, 140 mM NaCl and 0.05% Tween 20) containing 5% (w/v) BSA (FirstLink, UK) for 30 min. Cells were incubated with one of the primary antibodies, anti-HBcAg (Abcam, CA, USA), anti-HBsAg (Abcam), or anti-hepatitis B polymerase antibody (Santa Cruz Biotechnology, CA, USA) at 4°C overnight. Goat anti-mouse IgG antibody conjugated to Cy3 (Beyotime Institute of Biotech, China) was used as a secondary antibody. Post-staining images were taken with FLUOVIEW10-ASW laser scanning confocal microscope (Olympus, Japan).
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