Cells were harvested by scraping in an immunoprecipitation assay buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM sodium molybdate, and 0.5% Nonidet P-40) containing phosphatase and proteinase inhibitors (BestBioscience, Shanghai China). Lysates were clarified for 30 min at 13,000 × g, 4°C. A portion of the supernatants was incubated sequentially at 4°C overnight with protein A (Millipore) conjugated to an anti-HBx antibody (Abcam) or anti-HBc antibody (Abcam). Magnetic beads were washed three times with 200 μl of ice-cold immunoprecipitation assay buffer. Bound protein complexes and input fractions were examined by Western blot analysis using anti-human GATA-2 (Abcam) or anti-human GATA-3 (eBioscience) antibodies.
Anti hbx antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-HBx antibody is a laboratory reagent used for the detection and analysis of the HBx protein. It is a polyclonal antibody that specifically binds to the HBx protein, a regulatory protein encoded by the hepatitis B virus genome. The antibody can be used in various immunoassay techniques, such as Western blotting and immunohistochemistry, to study the expression and localization of the HBx protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Beyotime (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg alexafluor488, by Jackson ImmunoResearch (1 mentions)
Tcs sp5, by Leica (1 mentions)
Xbai enzyme, by New England Biolabs (1 mentions)
Pcmv vector, by Beyotime (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
3 protocols using anti hbx antibody
1
Immunoprecipitation of HBx and HBc Proteins
2
Immunofluorescence Localization of HBx and Hepsin
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Cells were plated on coverslips overnight at 37°C and then fixed with 4% formaldehyde at room temperature for 15 min. The cells were treated with 0.2% Triton X-100 in PBS on ice for 10 min and then washed 4 times with 500μl PBS. HBx was detected with anti-HBx antibody (Abcam, UK) and visualized with goat anti-rabbit IgG-Alexa Fluor 594 (Jackson, USA). Hepsin was detected with anti-V5 antibody (Invitrogen, USA) and visualized with goat anti-mouse IgG-Alexa Fluor 488 (Jackson, USA). 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Beyotime, China), was used for nuclear staining. Images were taken on a Confocal Laser Scanning Microscope (Leica TCS SP5, Germany).
3
Co-immunoprecipitation of YY1 and HBx
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pCMV-vector was bought from Beyotime technology (Shanghai, China). YY1 (HA tag in N terminal) and HBx (His tag in N terminal) sequences were constructed into pCMV-vectors through KpnI and XbaI enzyme (NEB, Ipswich, MA, USA) digestion. PCMV-HA-YY1 and pCMV-His-HBx vectors were co-transfected into Huh7 cells and co-IP assays were proceeded 2 days later. Briefly, 500μl cell lysis buffer (Beyotime, Shanghai, China) was used and then 1:50 diluted anti-HA (CST 3724, Danvers, MA, USA) or anti-His antibody (CST 12698, Danvers, MA, USA) was added into the solution at 4°C for overnight. Then magnetic beads were added and incubated for another 2 h. Later, beads were collected by magnetic separation rack and washed by lysis buffer mentioned above for five times. And finally, proteins were received by adding sample buffer to the beads with blending and incubating at 100°C for 30 min. The collected proteins were detected through western blots by anti-YY1 (CST 63227, Danvers, MA, USA) or anti-HBx antibody (Abcam ab2741, Cambridge, UK) in a concentration of 1:1000 dilution.
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