Horse anti-HBs (ab9193), rabbit anti-HBx (ab39716), mouse anti-HNF4α (ab181604), rabbit anti-actin (ab179467), and HRP-linked rabbit anti-horse IgG (ab6921) antibodies were purchased from Abcam (Cambridge, MA, USA). The rabbit anti-preS1 (10R-10460) antibody was purchased from Fitzgerald (Acton, MA, USA). The HRP-linked goat anti-rabbit IgG (BE0101-100) and HRP-linked goat anti-mouse IgG (BE0102-100) antibodies were purchased from Easybio (Beijing, China). The proteasome inhibitor MG132 (S2619) and lysosome inhibitor HCQ (S4430) were purchased from Selleck (Houston, TX, USA).
Ab9193
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab9193 is an anti-Ras antibody. It can be used to detect Ras proteins in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Axio observer z1, by Zeiss (3 mentions)
B0586, by Agilent Technologies (2 mentions)
Ab8639, by Abcam (2 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ab181604, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
Mg132 s2619, by Selleck Chemicals (1 mentions)
A5316, by Merck Group (1 mentions)
Pgc 1α, by Santa Cruz Biotechnology (1 mentions)
C ebpα, by Santa Cruz Biotechnology (1 mentions)
Sc 393668, by Santa Cruz Biotechnology (1 mentions)
Sc 365318, by Santa Cruz Biotechnology (1 mentions)
Hnf1α, by Santa Cruz Biotechnology (1 mentions)
Flag m2, by Merck Group (1 mentions)
Ab137055, by Abcam (1 mentions)
Monoclonal anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti flag antibody, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
12 protocols using ab9193
1
Antibody and Inhibitor Inventory
2
Protein Detection Antibodies in Hepatocyte Assays
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Various proteins were detected by the following antibodies: ISG20 (Abcam, Cambridge, UK, 1:2000), HBV core protein (B0586, Dako, Glostrup, Denmark, 1:2000), HBsAg (ab9193, Abcam, or Dako, 1:2000), hepatocyte nuclear factor 1α (HNF1α) (sc‐393668, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), FLAG‐M2 (A5316, 1:2000, Sigma), CEBPα (sc‐365318, 1:1000, Santa Cruz Biotechnology), HNF4α (sc‐374229, 1:2000, Santa Cruz Biotechnology), PGC‐1α (sc‐518025, 1:2000, Santa Cruz Biotechnology), and actin (A5316, 1:5000, Sigma).
3
Histopathological Evaluation of Viral Hepatitis
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Hepatic histopathologic changes were observed by hematoxylin and eosin (HE) staining. The location and expression of viral antigens in liver tissue were detected by IHC staining [21 (link)]. Paraformaldehyde-fixed paraffin-embedded tissue sections (4.5-μm thickness) were stained using HE and IHC. HBsAg and HBV core antigen (HBcAg) expression was determined in the liver sections by IHC using horse anti-HBsAg (1:1000) (ab9193, Abcam, Cambridge, UK) and rabbit anti-HBcAg (1:1000) (B0586, DAKO, Glostrup, Denmark). For the negative staining control, specimens were treated with normal saline only.
4
Monitoring HBsAg in HepG2-NTCP Cells
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HbsAg was monitored by IHC in HepG2-NTCP cells treated with HBV serum. After
transfection with NLRX1 overexpression plasmid or siRNA, cells were collected at
cell density of 5 × 106 cells/ml and added to sterile cell slippage
for 3 h of culture at 37 °C. The cells were processed with 4% paraformaldehyde
for 20 min, 0.5% Triton X-100 for 20 min, and 3% H2O2 for
15 min. After processing, cells were blocked with 5% normal goat serum
(Invitrogen, #16210072) for 2 h, primary antibodies including anti-HBs (1:50,
ab9193, Abcam, Cambridge, UK), anti-HBc (1:50, ab8639, Abcam) overnight at 4 °C,
and goat anti-mouse or rabbit IgG secondary antibody (1:500, Abcam) for 1 h.
After washing, cells were addressed with streptavidin-biotin-peroxidase (SABC,
Boster, Wuhan, China, #SA1020) at 37 °C for 20 min and colored with
diaminobenzidine hydrochloride (DAB, Boster, #AR1022). The color development
under the light microscope was observed and recorded.
transfection with NLRX1 overexpression plasmid or siRNA, cells were collected at
cell density of 5 × 106 cells/ml and added to sterile cell slippage
for 3 h of culture at 37 °C. The cells were processed with 4% paraformaldehyde
for 20 min, 0.5% Triton X-100 for 20 min, and 3% H2O2 for
15 min. After processing, cells were blocked with 5% normal goat serum
(Invitrogen, #16210072) for 2 h, primary antibodies including anti-HBs (1:50,
ab9193, Abcam, Cambridge, UK), anti-HBc (1:50, ab8639, Abcam) overnight at 4 °C,
and goat anti-mouse or rabbit IgG secondary antibody (1:500, Abcam) for 1 h.
After washing, cells were addressed with streptavidin-biotin-peroxidase (SABC,
Boster, Wuhan, China, #SA1020) at 37 °C for 20 min and colored with
diaminobenzidine hydrochloride (DAB, Boster, #AR1022). The color development
under the light microscope was observed and recorded.
5
Maintenance of Cell Lines for Protein Detection
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HepG2.2.15, HepAD38, Huh7, HEK293T, and Hela cells were maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM; HyClone, Utah, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO BRL, Grand Island, NY, USA), 1 mmol/L Na pyruvate, 100 μg/mL penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator.
The following antibodies and reagents were used: human ATP1B3 (#ab137055, Abcam, Cambridge, UK), β-tubulin (#RM2003, Ray Antibody Biotech, Beijing, China), myc antibody (#05-724, Millipore, Billerica, Massachusetts, USA), anti-flag mouse monoclonal antibody (#F1804, Sigma, New York, USA), anti-HBs (Ad/Ay) antibody (horse polyclonal, #ab9193, Abcam), and anti-V5 monoclonal antibody (#R960-25, Invitrogen). AP-conjugated anti-rabbit IgG antibody and AP-conjugated anti-mouse IgG antibody were purchased from Jackson (#115-055-062, #115-055-045, Lancaster, Pennsylvania, USA). All general chemicals were purchased from Sigma, Selleck and TAKARA unless otherwise stated.
The following antibodies and reagents were used: human ATP1B3 (#ab137055, Abcam, Cambridge, UK), β-tubulin (#RM2003, Ray Antibody Biotech, Beijing, China), myc antibody (#05-724, Millipore, Billerica, Massachusetts, USA), anti-flag mouse monoclonal antibody (#F1804, Sigma, New York, USA), anti-HBs (Ad/Ay) antibody (horse polyclonal, #ab9193, Abcam), and anti-V5 monoclonal antibody (#R960-25, Invitrogen). AP-conjugated anti-rabbit IgG antibody and AP-conjugated anti-mouse IgG antibody were purchased from Jackson (#115-055-062, #115-055-045, Lancaster, Pennsylvania, USA). All general chemicals were purchased from Sigma, Selleck and TAKARA unless otherwise stated.
6
Intracellular Localization of HBsAg and LC3
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Huh7 cells were seeded on cover slips and transfected with plasmids or siRNAs. Forty-eight hours after transfection, cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 10 min. Nuclei were stained with DAPI. HBsAg and LC3 were stained with Anti-Hepatitis B Virus Surface Antigen (Ad/Ay) antibody (ab9193, Abcam, UK) and LC3B (D11) XP® Rabbit mAb (3,868, Cell Signaling Technology, USA). Co-localization of SEL1L or LC3 (green) with HBsAg (red) was determined using a confocal microscope (LSM 710; Carl Zeiss) with objectives Plan-Apochromat 63×/1.40 oil Iris M27. Images were visualized by ZEN acquisition software (2012; Carl Zeiss) and analyzed by ImageJ.
7
Western Blot Analysis of Cellular Proteins
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Cells were seeded in 12-well plates and washed twice with PBS followed by lysing in 200 μl of Red Loading Buffer (Cell Signaling Technology). Ten microliters of the cell lysate were then resolved by electrophoresis in 12% SDS-PAGE, and proteins were transferred onto nitrocellulose filter membrane (GE Healthcare, USA). The membranes were blocked with 5% skim milk and incubated with antibodies against SEL1L (ab78298, Abcam, UK), EDEM1 (ab209660, Abcam, UK), HBsAg (ab9193, Abcam, UK), HBcAg (ab8639, Abcam, UK), LC3 (Cell Signaling Technology, USA), p62 (Cell Signaling Technology, USA), or β-actin (Cell Signaling Technology, USA). The membranes were washed with 1× TBST (as appropriate) and incubated with secondary antibodies (315-035-048, 111-035-045, Jackson ImmunoResearch, USA). Immunoreactive bands were captured by enhanced chemiluminescence system (RPN2106, GE Healthcare, USA).
8
ELISA for HBsAg and HBeAg Quantification
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ELISA was employed to analyze the levels of HBsAg and HBeAg using ELISA kits (cat. nos. INS1030201 and INS1030203; Huangshi Irons Biotechnology Co., Ltd.) according to the manufacturer's protocols. Antibodies against HBsAg (1:2,000; cat. no. ab9193; Abcam) and HBeAg (1:2,000; cat. no. ab91273; Abcam) were used for ELISA. The inhibitory rate was analyzed according to the formula: Inhibitory rate (%)=(Ccontrol-Ctested)/Ccontrol ×100%, where C is the concentration.
9
Immunofluorescence detection of HBsAg
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1 × 105 cells/well were cultured on collagen-coated glass coverslips and fixed the next day for 10 min in 4% paraformaldehyde. Cells were permeabilized for 30 min with 0.2% TritonX-100, followed by incubation with blocking solution (3% BSA, 10% FBS) for 1 h at RT, then labelled with an anti-HBsAg (1:150) from Abcam (ab9193) for 1 h at RT. Alexa Fluor® 488 AffiniPure Goat Anti-Horse IgG (H+L) (1:1000) (# 108-545-003) was incubated for 1 h at RT, followed by DAPI staining. Coverslips were then mounted on microscope slides using Prolong antifade reagent (ThermoFisher Scientific). Cells were analyzed using a confocal microscope (Zeiss LSM 780). Detector sensitivity was constant for all samples.
10
Immunofluorescence Assay for HBsAg
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Cells were washed twice in phosphate buffered saline (PBS), fixed in freshly prepared 3.7% paraformaldehyde in PBS for 15 min on ice, washed again three times, permeabilized in 0.1% Triton X‐100 in blocking solution (3% goat serum plus 0.5% bovine serum albumin [BSA] in PBS) for 30 min at room temperature, washed three times each for 5 min, and left in the blocking solution (3% goat serum plus 0.5% BSA in PBS) for 2 h. Cells were incubated overnight at 4°C with primary antibodies against HBsAg (ab9193; Abcam, Cambridge, MA). After washing with PBS, cells were incubated with FITC‐conjugated anti‐mouse secondary antibody at room temperature for 1 h. After washing with PBS three times, anti‐fade 4′,6‐diamidino‐2‐phenylindole (DAPI) (Wuhan Goodbio Biotechnology Co. Ltd., Wuhan, China) solution was added and images were captured using confocal laser‐scanning microscopy on a Nikon Digital ECLIPSE C1 system (Nikon Corporation, Tokyo, Japan).
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