Cells were washed three times using cold PBS and lysed in RIPA buffer with protease inhibitors. Approximate 0.03 mg of protein was separated with 10% SDS-PAGE gel and blotted into nitrocellulose membranes. Then membranes were blocked with 5% nonfat dried milk blocking buffer at room temperature for 1 h and incubated with diluted primary antibodies (1:1000) against GAPDH (#5174, Cell Signaling Technology, 1:1000), E-cadherin (ab15148, Abcam, 1:1000), α-catenin (ab51032, Abcam, 1:1000), Vimentin (#5741, Cell Signaling Technology, 1:1000), Fibronectin (ab2413, Abcam, 1:1000), VEGFA (ab51745, Abcam, 1:1000) and ANGPT2 (ab65835, Abcam, 1:1000) at 4 °C overnight. Then membranes were washed by TBST 3 times and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) at room temperature for 1 h. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, USA) α-catenin.
Ab51032
Manufactured by Abcam
Ab51032 is a laboratory reagent used for research purposes. It is a specific antibody that can be used to detect and identify a target protein or molecule in a sample. The core function of this product is to provide researchers with a tool for analyzing the presence and distribution of the target analyte in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Ab32084, by Abcam (2 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Ab2413, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab15148, by Abcam (1 mentions)
Ab51745, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti γ tubulin, by Merck Group (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti β catenin, by Thermo Fisher Scientific (1 mentions)
Anti gm130, by BD (1 mentions)
Ab10482, by Abcam (1 mentions)
Ab180593, by Abcam (1 mentions)
Fluoromouont g, by Southern Biotech (1 mentions)
Sp5 confocal scanning tandem rs, by Leica (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab214424, by Abcam (1 mentions)
6 protocols using ab51032
1
Western Blot Analysis of EMT Markers
2
Immunofluorescence Staining of Cytoskeletal and Cell-Cell Adhesion Proteins
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Cells were washed with 1x PBS and fixed for 20 min in paraformaldehyde (PFA) 4% at room temperature. After two washes with 1x PBS, they were permeabilized for 20 min in 1x PBS - 0.025% saponin and then blocked for 30 min in 1x PBS - 0.025% Saponin - 1% BSA (Bovine Serum Albumin, Sigma-Aldrich). Cells are incubated overnight at 4 °C in 1x PBS - 0.025% saponin, 1% BSA containing the primary antibody. The following day, after washing with 1x PBS, cells were incubated in 1x PBS - 0.025% saponin - 1% BSA containing the secondary antibody coupled to a fluorochrome for 1 h protected from light at room temperature. After two washes with 1x PBS, the nuclei were stained with 10 μg.ml−1 Hoechst33342 (H357C, Invitrogen) for 20 min. Slides were washed with 1x PBS, then with distilled water before being mounted on slides with Fluoromouont-G (0100-01, SouthernBiotech). The slides were visualized using an SP5 confocal scanning Tandem RS (Leica) and analyzed by ImageJ. The following antibodies were used: anti-γ-tubulin (T6557, Sigma-Aldrich); anti-GM130 (610823, BD Biosciences); anti-E-cadherin (ab1416, Abcam); anti-E-cadherin (610181, BD Biosciences); anti-Galectin-7 (ab10482, Abcam); anti-β-catenin (MA1-301, Thermo Scientific); anti-α-catenin (13-9700, Thermo Scientific); anti-α-catenin (ab51032, Abcam); anti-S100A11 (ab180593, Abcam).
3
Western Blot Analysis of EMT and Angiogenesis Markers
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Hep3b cells were washed in PBS and lysed using the protein extraction reagent RIPA (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of proteins was measured by BCA kit (cat. no. ab102536; Abcam). Equivalent amounts of proteins (30 µg) from each sample were electrophoresed on SDS-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, blocked in 4% skim milk for 2 h at room temperature and incubated with the following specific primary antibodies: E-cadherin antibody (ab219332 1:1,000; Abcam), α-catenin antibody (ab51032 1:2,000; Abcam), N-cadherin (ab76011, 1:5000 dilution, Abcam), vimentin (ab92547 1:1,000; Abcam), p-PI3K (ab182651 1:1,000; Abcam), PI3K (ab227204 1:1,000; Abcam), p-AKT (ab38449, 1:500 dilution, Abcam), AKT (ab18785 1:1,000; Abcam), VEGF (ab214424 1:1,000; Abcam), VEGFR2 (ab221679 1:1,000; Abcam), Snail (ab53519 1:1,000; Abcam), Slug (ab27568 1:1,000; Abcam) and MPP9 (ab38898 1:1,000; Abcam) overnight at 4°C. β-actin (ab8277 1:1,000; Abcam) was used as internal reference. Then, the membranes were incubated in HRP-linked goat anti-rabbit IgG secondary antibody (ab97051; 1:10,000; Abcam) for 2 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an ECL substrate buffer (EMD Millipore) and membranes were scanned with Gel Doz EZ imager (Bio-Rad Laboratories, Inc.).
4
Investigating CTNNA1-LC3B Interaction
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Binding of LC3B (purified from E. coli) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.
5
Western Blot Analysis of EMT Markers
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Samples were lysed in RIPA lysis buffer (Thermo Scientific) supplemented with Complete protease inhibitor cocktail (Roche) and protein concentrations were measured using the detergent-compatible Protein Assay Kit (Bio-Rad). Samples were then mixed with Laemmli sample buffer, boiled at 95°C for 5 min, resolved by SDS–PAGE, and transferred to nitrocellulose membranes. Membranes were then blocked in 5% nonfat milk in 0.1% Tween-20 in Tris-buffered saline and incubated overnight at 4°C in blocking buffer containing antibodies specific for ILK (3862, 1:1000; Cell Signaling), E-cadherin (3195, 1:1000; Cell Signaling), vimentin (V5255, 1:1000; Cell Signaling), Snail (3895, 1:500; Cell Signaling), αSMA (A5228, 1:500; Sigma), FAK (3285S, 1:500; Cell Signaling), pY397-FAK (44625G, 1:500; Invitrogen), paxillin (ab32084, 1:1000; Abcam), pY118-paxillin (44722G, 1:1000; Invitrogen), α-catenin (ab51032, 1:3500; Abcam), β-catenin (ab32572, 1:3500; Abcam), or GAPDH (3683S, 1:1000; Cell Signaling). Bands were detected using horseradish-peroxidase–conjugated secondary antibodies (1:5000; Cell Signaling) and Amersham ECL Western Blotting Reagent (GE Healthcare) as a chemiluminescent substrate. Densitometry analysis was performed using ImageJ.
6
Immunofluorescence Characterization of Epithelial-Mesenchymal Transition
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Samples were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and then permeabilized with 0.3% Triton X-100 for 30 min. After blocking for 1 h with 5% goat serum (Sigma) and 2% bovine serum albumin, the samples were incubated with primary antibody against E-cadherin (3195, 1:300; Cell Signaling), Snail (3895, 1:300; Cell Signaling), αSMA (A5228, 1:200; Sigma), ZO1 (40-2200, 1:300; Thermo Fisher Scientific), vimentin (V2258, 1:200; Sigma), cleaved caspase-3 (9961, 1:200; Cell Signaling), FAK (ab40794, 1:200; Abcam), pY397-FAK (44-625G, 1:50; Invitrogen), paxillin (ab32084, 1:200; Abcam), pY118-paxillin (44-722G, 1:200; Invitrogen), α-catenin (ab51032, 1:200; Abcam), or β-catenin (D10A8, 1:100; Cell Signaling). Samples were then washed with PBS and incubated with Alexa Fluor–conjugated secondary antibodies (1:200; Invitrogen). Nuclei were counterstained with Hoechst 33342 (1:1000; Invitrogen). After additional washes with PBS, samples were visualized using a 20×/0.45 NA air objective on a Nikon Eclipse Ti-U inverted fluorescence microscope (Nikon) equipped with a Hamamatsu ORCA charge-coupled device camera. Image analysis was performed on at least 200 cells for each experimental group over three independent experiments using ImageJ.
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