RIPA (GBCBIO, G3424) was used to lyse cells and extract total protein to prepare protein samples. The protein lysates were separated by conventional electrophoresis, transferred onto the membrane, and washed with PBS, followed by blocking with 1% BSA for 2 h. Then, primary antibodies Box (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab32124, Abcam, Cambridge, UK), Caspase-1 (ab32503, Abcam, Cambridge, UK), Cleaved Caspase-3 (ab32503, Abcam, Cambridge, UK), Caspase-8 (ab207802, Abcam, Cambridge, UK), Caspase-9 (ab2302, Abcam, Cambridge, UK), Caspase-11 (ab25901, Abcam, Cambridge, UK), Cyclin D1 (ab202068, Abcam, Cambridge, UK), ERK 1/2 (ab180673, Abcam, Cambridge, UK), p-ERK 1/2 (ab16663, Abcam, Cambridge, UK), WNT (ab17942, Abcam, Cambridge, UK), β-catenin (ab223500, Abcam, Cambridge, UK), GSDMD (ab15251, Abcam, Cambridge, UK) and β-actin (ab32572, Abcam, Cambridge, UK) (abcam, ab32503, ab32124, ab207802, ab2302, ab25901, ab202068, ab180673, ab16663, ab17942, ab223500, ab15251, ab32572, ab219800, ab8227) each at 1:1000 dilution were incubated with the membrane overnight at 4°C. The next day, HRP (ab32572, Abcam, Cambridge, UK) and labeled IgG secondary antibody (ab150077, Abcam, Cambridge, UK) each at 1:5,000 were further incubated with the membrane at room temperature for 1 h. ECL Luminescence Kit (Thermo, 32209) was used to develop the signal in a dark room.
Ab32124
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Japan
Ab32124 is a protein-based lab equipment product. It is designed for use in research applications. The core function of this product is to facilitate the study and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (253 mentions)
Ab8245, by Abcam (59 mentions)
Ab32042, by Abcam (56 mentions)
Ab2302, by Abcam (51 mentions)
Pvdf membrane, by Merck Group (51 mentions)
Ab9485, by Abcam (40 mentions)
Ab6721, by Abcam (38 mentions)
Ab205718, by Abcam (34 mentions)
Ab13847, by Abcam (34 mentions)
Ripa lysis buffer, by Beyotime (32 mentions)
Ab8226, by Abcam (27 mentions)
Ab8227, by Abcam (26 mentions)
Ab181602, by Abcam (24 mentions)
Ripa buffer, by Beyotime (24 mentions)
Ab2324, by Abcam (23 mentions)
Ab32351, by Abcam (22 mentions)
Bca protein assay kit, by Beyotime (20 mentions)
Ab182733, by Abcam (20 mentions)
Ab40772, by Abcam (18 mentions)
Ab134175, by Abcam (16 mentions)
397 protocols using ab32124
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Cell Signaling
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Total proteins were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) and protein concentration was quantified with a BCA protein assay kit (Beyotime, Shanghai, China). Protein samples were isolated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against SGK1 (Abcam, ab32374, 1:500), Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab243140, 1:1000), Cleaved caspase-3 (Abcam, ab2302, 1:1000), caspase-3 (Abcam, ab184787, 1:2000), Cleaved caspase-9 (Abcam, ab2324, 1:1000), caspase-9 (Abcam, ab32539, 1:5000), GRP78 (Abcam, ab108615, 1:10,000), p-PERK (CST company, 3179s, 1:1000), PERK (CST company, 3192s, 1:1000), ATF4 (Abcam, ab184909, 1:1000), CHOP (Abcam, ab11419, 1:1000) and GAPDH (Abcam, ab32124, 1:1000) and incubated with the corresponding secondary antibody (Abcam, ab205718, 1:50,000) for 1.5 h at room temperature. Protein signals were visualized using electrochemiluminescence (ECL; Beyotime, Shanghai, China) method and detected by a Bio-Rad imaging system (Bio-Rad, CA, USA).
3
Western Blot Analysis of Apoptosis and Autophagy Markers
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HUVECs were lyzed using RIPA lysis buffer (Beyotime, Shanghai, China) and cell supernatants were collected by centrifugation. The protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were isolated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies against Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab243140, 1:500), Cleaved caspase-3 (Abcam, ab2302, 1:500), Caspase-3 (Abcam, ab184787, 1:2000), Beclin-1 (Abcam, ab207612, 1:2000), Atg5 (Abcam, ab108327, 1:10,000), p62 (Abcam, ab207305, 1:1000), GRP78 (Abcam, ab108615, 1:10,000), CHOP (Abcam, ab11419, 1:1000), Caspase-12 (Abcam, ab62484, 1:2000), XBP-1 (Abcam, ab220783, 1:1000) and GAPDH (Abcam, ab32124, 1:1000) overnight at 4°C. Membranes were washed in TBST, followed by incubation with secondary antibodies (Abcam, ab205718, 1:50,000; Abcam, ab205719, 1:20,000) for 1 h at room temperature. Protein bots were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China).
4
Proteomic Analysis of Cell Signaling Molecules
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The hEM15A cells were lysed in RIPA buffer (Sigma, USA) containing protease inhibitor cocktail (Sigma). Then, proteins were isolated using 10% SDS-PAGE gels, transferred onto PVDF membranes (Millipore, USA), and blocked with nonfat milk (5%) at room temperature for 2 h. Afterward, the membranes were incubated with specific primary antibodies against CDK2 (ab32147, 1:1000; Abcam, USA), CDK4 (ab137675, 1:1000), CDK6 (ab124821, 1:1000), Cyclin D1 (ab16663, 1:1000), Bcl-2 (ab32124, 1:1000), Bax (ab32124, 1:1000), cleaved Caspase-3 (ab4051, 1:1000), N-cadherin (ab18203, 1:1000), vimentin (ab137321, 1:1000), E-cadherin (ab15148, 1:1000), TMSB4X (ab167650, 1:1000), or TGF-β2 (ab113670, 1:1000) at 4°C overnight and incubated with corresponding HRP-conjugated secondary antibodies (ab131368 and ab191866; both 1:5,000) for 2 h at room temperature. Eventually, the proteins were visualized via ECL (Thermo Pierce), with GAPDH as the loading control.
Bioinformatics analysis
StarBase website (http://starbase.sysu.edu.cn ) was used to find candidate microRNAs (miRNAs) for circPIP5K1A, and miRmap (https://mirmap.ezlab.org ), PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html ), and PicTar (http://www.pictar.mdc-berlin.de ) databases were used to predict the candidate target genes for miR-153-3p.
Bioinformatics analysis
StarBase website (
5
Western Blot Analysis of Apoptosis Regulators
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Radioimmunoprecipitation assay lysis buffer (Sangon) was applied for total protein extraction, followed by Western blot detection referring to the issued description.16 (link)
The primary Abs targeting B-cell lymphoma-2 (Bcl-2; ab32124, 1:1000), Bcl-2-associated X (Bax; ab182733, 1:1000), RORA (ab256799, 1:1000), GAPDH (ab181603, 1:1000) were provided by Abcam (Cambridge, UK). Goat Anti-rabbit IgG H&L (HRP) secondary Ab (Abcam, ab205718, 1:5000) was incubated at 25°C for 1 h, followed by analysis of protein bands through ECL luminescence reagent (Sangon) and Image J software (NIH, Bethesda, MD, USA).
The primary Abs targeting B-cell lymphoma-2 (Bcl-2; ab32124, 1:1000), Bcl-2-associated X (Bax; ab182733, 1:1000), RORA (ab256799, 1:1000), GAPDH (ab181603, 1:1000) were provided by Abcam (Cambridge, UK). Goat Anti-rabbit IgG H&L (HRP) secondary Ab (Abcam, ab205718, 1:5000) was incubated at 25°C for 1 h, followed by analysis of protein bands through ECL luminescence reagent (Sangon) and Image J software (NIH, Bethesda, MD, USA).
6
Protein Isolation and Western Blot Analysis
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Total proteins were isolated using RIPA buffer and separated via 10% SDS-PAGE. These proteins were then transferred to PVDF membranes, which were blocked with 5% skimmed milk to prevent non-specific binding. Then, the membranes were incubated with the primary antibodies at 4 °C overnight and the secondary antibody for 1 h at room temperature. The antibodies were PCNA (ab18197; 1:1000; Abcam, Cambridge, UK), Bax (ab32503; 1:1000; Abcam), FABP4 (ab66682; 1:1000; Abcam), Bcl-2 (ab32124; 1:1000; Abcam), Sox2 (ab97959; 1:1000; Abcam), Oct4 (ab19857; 1:1000; Abcam), ALDHA1 (15910-1-AP; 1:1000; Proteintech, Wuhan, China), Glut1 (ab15309; 1:1000; Abcam), LDHA (ab52488; 1:1000; Ab-cam), p-ERK (4370; 1:1000; Cell Signaling Technology, Boston, MA, USA), ERK (4695; 1:1000; Cell Signaling Technology), p-mTOR (5536; 1:1000; Cell Signaling Technology), mTOR (2983; 1:1000; Cell Signaling Technology), and GAPDH (ab181602; 1:1000; Abcam). Finally, the enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) was conducted to obtain the combined protein signals.
7
Western Blot Analysis of Signaling Proteins
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Total cell protein was extracted using a radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) according to the manufacturer's protocol. The concentration of protein was determined using a BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg/lane) were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with 5% non-fat milk at 4°C for 2 h, and incubated overnight at 4°C with primary antibodies (anti-PI3K, 1:1,000, ab151549; anti-Akt, 1:10,000, ab179463; anti-PTEN, 1:10,000, ab32199; anti-Bcl-2 1:1,000, ab32124; anti-Bax, 1:5,000, ab32503; and anti-β-actin, 1:200, ab115777; all purchased from Abcam, Cambridge, MA, USA). The following day, the membranes were washed and incubated at room temperature with the horseradish peroxidase-conjugated secondary antibody (1:1,000; A0208; Beyotime Institute of Biotechnology) for 45 min. Finally, membranes were incubated with BeyoECL Plus (Beyotime Institute of Biotechnology), and detected using a ChemiDoc™ XRS+imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). -actin was applied as the internal control.
8
Western Blot Analysis of Apoptosis and EMT Markers
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The GC cells was separated using RIPA lysate (89901, Thermo Fisher Scientific, Inc.), and the protein concentration was determined using a Protein Assay kit (A53227, Thermo Fisher Scientific, Inc.). A volume of 30 µg proteins of each tissue was separated on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane (HVLP04700, EMD Millipore), which the membrane was then washed with TBS and blocked with 5% non-fat milk at 37°C for 1 h and shaken for 2 h at room temperature. The membrane was then incubated with the following primary antibodies: Anti-Bcl2 (1:1,000; 26 kDa; rabbit; ab32124), anti-procaspase-3 (1:10,000; 35 kDa; rabbit; ab32499), anti-cleaved caspase-3 (1:500; 17 kDa; rabbit; ab32042), Vimentin (1:1,000; 54 kDa; rabbit; ab92547), N-cadherin (1 µg/ml; 130 kDa; rabbit; ab18203), E-cadherin (1:10,000; 97 kDa; rabbit; ab40772) or GAPDH (1:10,000; 36 kDa; rabbit; ab181602) (all from Abcam) overnight at 4°C. The target band was incubated with a goat anti-rabbit IgG H&L (HRP) (1:5,000; ab205718; Abcam) for 2 h at room temperature. Finally, the signals were measured by ECL reagent (6883, Cell Signaling Technology, Inc.), and the gray values of the bands were analyzed and counted using ImageJ software (version 5.0, Bio-Rad Laboratories, Inc.) (16 (link)).
9
Caspase Activation Immunoblotting Assay
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The cells seeded on the plates were transfected with indicated plasmids. Five hours before the collection, cells were treated with staurosporine (1 μM, Sigma) or dimethyl sulfoxide and then were lysed for Western blot. The indicated primary antibodies were used to detect the full-length and cleaved caspase3/8/9. Antibodies used in immunoblotting are as follows: anti-caspase3 (abcam, ab32351), anti-caspase8 (proteintech, 13423-1-AP), anti-caspase9 (proteintech, 10380-1-AP), anti-Bcl-2 (abcam, ab32124), and anti-BAX (abcam, ab32503).
10
Protein Expression Analysis in Colorectal Cancer
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After cultivation for 48 h, transfected colorectal cancer cells were collected and lysed with RIPA lysis buffer (Beyotime, Shanghai, China). Then, the quality and quantity of total proteins were measured with a bicinchoninic acid (BCA) protein assay kit (Cowbiotech, Beijing, China). Subsequently, total protein samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). Next, the blocking experiment was conducted by adding 5% bovine serum albumin (BSA) into the membranes, and then the membranes were incubated overnight at 4°C with primary antibodies against proliferating cell nuclear antigen (PCNA) (ab29; Abcam, Cambridge, MA) (1 μg/ml), B-cell lymphoma 2 (Bcl-2) (ab32124; Abcam) (1/1,000), E-cadherin (E-cad) (ab1416; Abcam) (1/50), matrix metalloprotein 9 (MMP9) (ab38898; Abcam) (1/1,000), HMGA2 (ab97276; Abcam) (1/1,000), or GAPDH (ab37168; Abcam) followed by incubation with a horseradish peroxidase (HRP)-conjugated second antibody (ab6721; Abcam), and the bands were scanned using Image Lab software (Bio-Rad).
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