Transfected PC3 or DU145 cells were dissolved by radioimmunoprecipitation Assay lysis solution (Thermo Fisher Scientific). The protein samples were separated using 10% SDS‐PAGE (Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) (Bio‐Rad Laboratories), transferred into polyvinylidene fluoride (PVDF) membranes (Millipore) afterwards. The membranes were blocked and cultured with primary antibodies, including anti‐E‐cadherin antibody (1/50, ab1416, Abcam, Cambridge, USA), anti‐N‐cadherin antibody (1/1000, ab76057, Abcam), anti‐Bax antibody (1/10000, ab32503, Abcam), anti‐Bcl‐2 antibody (1/2000, ab182858, Abcam), anti‐SMURF1 antibody (1/200, ab38866, Abcam) and anti‐GAPDH antibody (1/2000, ab245357, Abcam). Next day, the membranes were incubated with a secondary antibody after washed thrice. Protein bands were quantified by Odyssey CLx v2.1 software (Media Cybernetics). GAPDH was considered as a loading control.
Ab245357
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab245357 is a primary antibody designed for the detection of a specific target protein. It is suitable for use in various laboratory techniques, such as immunohistochemistry and western blotting. The core function of this product is to bind to and enable the identification of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab38866, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis solution, by Thermo Fisher Scientific (1 mentions)
Ab76057, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ab8978, by Abcam (1 mentions)
Ab76055, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab233222, by Abcam (1 mentions)
Proteojet mammalian cell lysis reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Protease inhibitor and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Ab22609, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
4 protocols using ab245357
1
Evaluating EMT and Apoptosis Markers
2
Western Blot Analysis of Cellular Proteins
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Total cell lysates from cells were prepared using ProteoJET™ Mammalian Cell Lysis
Reagent (Thermo Scientific, Waltham, MA, USA). BCA Protein Assay Kit (Thermo
Scientific) was used to quantify protein concentration. Proteins (50 μg) were
separated using SDS-PAGE (10%), transferred to nitrocellulose membranes
(162-0115; Bio-Rad Laboratories), and blocked in Tris-buffered saline with
Tween-20 (TBST) containing 5% non-fat milk. The membranes were probed with the
specific primary antibodies. After being washed in TBST 3 times, the membranes
were incubated with corresponding horseradish peroxidase-conjugated secondary
antibody (ab205718 or ab6728, 1:5000). Finally, protein bands were visualized
using the enhanced chemiluminescent substrate, according to the manufacturer’s
protocol (Thermo Scientific). The specific antibodies to E-cadherin (1:1000,
ab76055), N-cadherin (1:1000, ab18203), Vimentin (1:1000, ab8978), SUMO2
(1:1000, ab233222), SUMO3 (1:1000, ab203570) and GAPDH (1:2000, ab245357) were
purchased from Abcam (Cambridge, UK).
Reagent (Thermo Scientific, Waltham, MA, USA). BCA Protein Assay Kit (Thermo
Scientific) was used to quantify protein concentration. Proteins (50 μg) were
separated using SDS-PAGE (10%), transferred to nitrocellulose membranes
(162-0115; Bio-Rad Laboratories), and blocked in Tris-buffered saline with
Tween-20 (TBST) containing 5% non-fat milk. The membranes were probed with the
specific primary antibodies. After being washed in TBST 3 times, the membranes
were incubated with corresponding horseradish peroxidase-conjugated secondary
antibody (ab205718 or ab6728, 1:5000). Finally, protein bands were visualized
using the enhanced chemiluminescent substrate, according to the manufacturer’s
protocol (Thermo Scientific). The specific antibodies to E-cadherin (1:1000,
ab76055), N-cadherin (1:1000, ab18203), Vimentin (1:1000, ab8978), SUMO2
(1:1000, ab233222), SUMO3 (1:1000, ab203570) and GAPDH (1:2000, ab245357) were
purchased from Abcam (Cambridge, UK).
3
Quantification of CAMK2A in Mouse Spinal Cord
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The L4-L6 spinal cord segments of each mouse were removed and treated with lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitor and phosphatase inhibitor cocktail (Sigma-Aldrich; Merck KGaA). BCA assay was used to detect the amount of protein. Proteins extracted from the spinal cord (40 µg) were separated via 10% SDS-PAGE followed by transfer onto PVDF membranes (EMD Millipore) and incubation with 5% non-fat dry milk in TBS containing 0.3% Tween-20 buffer for 2 h at room temperature. Subsequently, the blots were incubated with primary antibodies against CAMK2A (1:500, ab22609, Abcam) and GAPDH (1:1,000, ab245357, Abcam) at 4°C for 12 h, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:1,000; cat. no. 6927-100, Amyjet) for another 2 h at room temperature. Finally, the blots were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). The bands were semi-quantified using ImageJ software V1.8.0 (National Institutes of Health).
4
Western Blot Analysis of Osteogenic Markers
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The cells were washed three times with PBS and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors for 30 min at 4 C. For the Western blot analysis, 15 μL of the sample was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes. The primary antibodies used were rabbit polyclonal anti-GCN5 (1:1000, ab231075, Abcam, China); rabbit monoclonal anti-Axin-2 (1:1000, ab109307, Abcam, China); rabbit monoclonal anti-β-catenin (1:1000, ab32572, Abcam, China) and mouse monoclonal phosphorylated β-catenin (1:500, 05-665-AF647, Millipore, United States); rabbit monoclonal anti-H3K9ac (1:1000, ab10812, Abcam, China); rabbit monoclonal anti-RUNX2 (1:1000, ab192256, Abcam, China); rabbit monoclonal anti-OCN (1:1000, ab102936, Abcam, China); and rabbit monoclonal anti-OPN (1:1000, ab63856, Abcam, China). For normalization of protein loading, GAPDH antibody (1:1000, ab245357, Abcam, China) was used. The protein bands were measured with ImageJ 1.48v software and normalized to the corresponding GAPDH bands. The relative density of each target protein normalized to the control was used to represent the changes in expression of target proteins.
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