For the immunofluorescence assay, cells were plated on glass cover slips and stained as described below. Cells were fixed with 4% formaldehyde for 15 min and then incubated in Triton extraction buffer (300 mM sucrose, 20 mM HEPES pH7.9, 50 mM NaCl, 3 mM MgCl, 0.5% Triton X-100) for 30 min to increase the permeability of cell membrane. Next, the cells were blocked with 2% BSA for 40 min and incubated with rabbit anti-HBc polyclonal antibody (DAKO) at 4°C in wet box overnight. An FITC-conjugated anti-rabbit antibody was used as secondary antibody and DAPI was used to stain nuclei. Finally, confocal microscope was used to observe the images.
Rabbit anti hbc polyclonal antibody
Manufactured by Agilent Technologies
Sourced in Denmark
The Rabbit anti-HBc polyclonal antibody is a laboratory product used for the detection and analysis of the hepatitis B core antigen (HBcAg). It is a polyclonal antibody raised in rabbits against the HBcAg, and can be used in various immunoassay techniques to identify and quantify the presence of HBcAg in samples.
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5 protocols using rabbit anti hbc polyclonal antibody
1
Immunofluorescence Staining of HBc Antigen
2
HBV Capsid Analysis via Phos-tag SDS-PAGE
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HBV capsids were mixed with 5X SDS-loading dye (5% β-Mercaptoethanol, 0.02% Bromophenol blue, 30% Glycerol, 10% SDS, 250 mM Tris-HCl, PH 6.8) and boiled for 10 min before loading onto polyacrylamide-bound Mn2+-Phos-tag SDS-PAGE (Wako Pure Chemical Industries, Richmond, VA, USA)62 (link)63 64 (link). After electrophoresis, the gel was soaked for 10 min in protein transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) containing 1 mM EDTA, followed by soaking for another 10 min in the same buffer without EDTA. Western blot analysis was performed by using rabbit anti-HBc polyclonal antibody (DAKO, Glostrup, Denmark).
3
Native Agarose Gel Electrophoresis and Immunoblot
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Experimental procedures for native agarose gel electrophoresis and immunoblot are as described elsewhere48 (link). Rabbit anti-HBc polyclonal antibody (DAKO, Glostrup, Denmark) was used in 1:2000 dilutions, while goat anti-rabbit-IgG-HRP antibody (GeneTex Inc, Taiwan) was used in 1:4000.
4
Western Blot Analysis of Protein Samples
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HCC cell lines in a well of 60×15mm Dish or tissue samples were lysed 300 μl RIPA Lysis Buffer (Beyotime, China) supplemented with 1mM Phenylmethanesulfonyl fluoride. The protein concentration was determined using the Bradford assay. 20 μg or 50 μg of total protein was electrophoretically separated on 12% SDS-PAGE gel and transferred from the gel onto a PVDF membrane (Millipore, USA). Western blotting was performed according to standard procedures. PVDF membranes were washed with TBST containing NaCl, Tris-HCl, and Tween-20 and incubated with primary antibodies against target proteins at 4 ℃ overnight, followed by five washes with TBST. PVDF membranes were incubated with the appropriate secondary antibodies at room temperature for 1-2 h and washed five times with TBST. Finally the membranes were visualized by chemiluminescence with an ECL kit (Biorad, USA). Antibodies against Id1, E2F4, GFP, Cherry, GST, His, GAPDH and β-actin obtained from Santa Cruz Biotechnology (USA). Rabbit anti-HBc poly-clonal antibody (B0586) was obtained from Dako (Denmark).
5
Intrahepatic HBc Protein Immunohistochemistry
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Immunohistochemical staining for intrahepatic core protein was performed on paraffin-embedded liver tissue sections as described. Briefly, formalin-fixed and paraffin-embedded specimens from mice liver were sectioned into 5-um-thick slices. After deparaffinization, the slides were incubated with rabbit anti-HBc polyclonal antibody (Dako) at 4°C overnight, and detected with diaminobenzidine (DAB) staining. Finally, the images were captured by microscope.
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