The concentration of total proteins was validated using a BCA protein detection kit (Beyotime, China). A precise amount of total protein, 40 μg for each sample, was loaded for performing 2-h sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the proteins, driven by 150 mA were continuously transferred onto the PVDF membrane for 2 hrs. With 50 g/L skim milk adopted to block proteins for 2 hrs, the rabbit anti-human primary antibodies (Abcam, USA) for E-cadherin (1:500, Catalog no.: ab15148), N-cadherin (1:1000, Catalog no.: ab76057), Vimentin (1:1500, Catalog no.: ab137321), CHK1 (1:10,000, Catalog no.: ab40866) and GAPDH (1:10,000, Catalog no.: ab181602) were prepared to incubate proteins, which was then maintained at 4°C for overnight. After washing the membrane with 1×Tris buffered saline Tween for 10 mins, corresponding secondary antibodies tabbed by horseradish peroxidase (goat anti-rabbit, 1:3000, Catalog no.: ab97051) were supplemented to incubate proteins for 1 hrs. In addition, electrochemiluminescence agent (Millipore, Billerica, MA, USA) was added onto the membrane for development, and gray values of protein bands were quantified by Image-Pro Plus software, with GAPDH designated as the internal reference.
Rabbit anti human primary antibody
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-human primary antibodies are laboratory reagents used in various immunodetection techniques, such as Western blotting, ELISA, and immunohistochemistry. These antibodies are raised in rabbits and specifically target human proteins or antigens, allowing for the detection and analysis of their expression or localization in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein detection kit, by Beyotime (1 mentions)
Alcian blue 8gx, by Carl Roth (1 mentions)
Gapdh, by Abcam (1 mentions)
Cyclin a2, by Abcam (1 mentions)
Tgf β, by Abcam (1 mentions)
C jun, by Abcam (1 mentions)
C fos, by Abcam (1 mentions)
Trans blot sd semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Goat anti rabbit secondary antibody, by Abcam (1 mentions)
4 protocols using rabbit anti human primary antibody
1
Protein Expression Analysis by Western Blot
2
Characterization of Cartilage Markers
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After two weeks of induction, the pellets were fixed in cold 4 % paraformaldehyde, embedded in paraffin, sectioned (5 µm thick) and used for immunocytochemistry and Alcian Blue staining. After deparaffinization and rehydration, antigen retrieval was carried out using sodium citrate. The immunostaining was carried out using type II Collagen (Col-II, 1:20 diluted) or type X Collagen (Col-X, 1:80 diluted) rabbit anti-human primary antibodies (Abcam) at 4 °C overnight and stained with PE-conjugated goat anti-rabbit secondary antibody (1:100 diluted, 1 h at 37 °C, eBioscience). For GAG staining, the sections were stained with Alcian Blue 8GX (Roth) as previously described (Shafiee et al., 2011[36 (link)]). Sections were then photographed using a fluorescent microscope (Nikon) assisted with an IP-camera (Genucam).
3
Western Blot Analysis of Protein Expression
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Cells were collected and lysed with protease and phosphatase inhibitors. The protein concentration of the samples was determined using a BCA Protein Analysis Kit (Sangon Biotech, Shanghai, China). The proteins in the sample (20 μL) were separated by electrophoresis on 12% SDS-PAGE gel and proteins were transferred to a PVDF membrane using a semi-dry transfer system (Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell; Bio-Rad). The membrane was blocked with 5% non-fat dry milk for 2 h and then incubated overnight at 4 °C with rabbit anti-human primary antibody (Abcam, Cambridge, UK), GAPDH (1:2000), FGF1 (1:2000), p53 (1:1000), C-FOS (1:2000), C-JUN (1:2000), TGFβ (1:2000), and cyclin A2 (1:2000) antibodies (Abcam, Cambridge, UK). After washing with TBST three times, the membrane was incubated with HRP-labelled anti-rabbit secondary antibody (Abcam) for 2 h at 37 °C. Finally, ECL luminescence was used to visualise the protein bands. GAPDH was the internal reference.
4
Immunohistochemical Analysis of Prostate Cancer
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The surgically removed tissue samples were dehydrated in accordance with the dehydration protocol using an automated dehydrator after already being fixed with 4% paraformaldehyde. Prostate cancer samples were completely embedded in paraffin and sliced into 5-µm-thick sections. Slices were baked at 60 degrees for 1 h, followed by dewaxing and hydration. After antigen retrieval and removal of endogenous peroxidase, nonspecific epitopes were blocked by immunostaining blocking solution at room temperature. Sections were stained overnight with rabbit anti-human primary antibody (Abcam, Cambridge, UK), and sections were stained with goat anti-rabbit secondary antibody (Abcam, Cambridge, UK). Hematoxylin and 3,3-diaminobenzidine (DAB) were utilized to stain tissue sections separately. After immersing the sections in various concentrations of graded ethanol, they were covered with coverslips. The sections were observed and photographed using a microscope.
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