Total protein was extracted from tissues or cells by use of lysis buffer. Bradford method was used to determine the concentration of proteins. Aliquots supernatant proteins were added with loading buffer and subjected to 10% SDS/PAGE. The resolved proteins were transferred to PVDF membranes (Beyotime, Shanghai, China) and 5% milk with 0.1% Triton X-100 blocked the membranes, and then samples incubated with different primary antibodies: rabbit anti-CCNE1 antibody (ab3927, 1:1000, Abcam, U.S.A.), anti-Cyclin A antibody (sc-53228, 1:1000, Santa Cruz, U.S.A.), anti-Cyclin D1 antibody (ab134175, 1:10000, Abcam, U.S.A.), anti-C-myc antibody (ab32072, 1:1000, Abcam, U.S.A.), anti-Bax antibody (ab182734, 1:1000, Abcam, U.S.A.), anti-Bcl-2 antibody (ab182858, 1:2000, Abcam, U.S.A.) and anti-GAPDH (ab8245, 1:2000, Abcam, U.S.A.) for overnight at 4°C. Blots were then incubated with HRP–conjugated goat anti-Rabbit IgG (Proteintech, U.S.A.) as secondary antibodies. Membranes were washed with TBS three times for 5 min and the blots were viewed with ECL (Thermo Fisher Scientific, Inc., U.S.A.). The quantitation of the relative expression of protein was performed using Quantity one (Bio-Rad, U.S.A.).
Ab182734
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab182734 is a laboratory product manufactured by Abcam. It is a piece of equipment designed for use in scientific research and analysis. The core function of this product is to facilitate specific tasks or procedures within a laboratory setting. No further details about its intended use or capabilities can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
Ab182858, by Abcam (5 mentions)
Ab6721, by Abcam (5 mentions)
Ab194583, by Abcam (4 mentions)
Ab184787, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab8227, by Abcam (3 mentions)
Ab181602, by Abcam (3 mentions)
Ab8245, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Ab32042, by Abcam (2 mentions)
Ab49822, by Abcam (2 mentions)
Ab194726, by Abcam (2 mentions)
Ab16502, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab3927, by Abcam (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Ab134175, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
19 protocols using ab182734
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Cell Proliferation and Apoptosis Markers
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Cells from the various treatment groups were lysed using radio-immunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Protein determination by was performed via the BCA method and total protein was isolated. Equal amounts (25 µg) total protein were separated on a 10% gel via sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% milk for 1 h at room temperature. The PVDF membrane was then washed with TBST (0.1% Tween-20) three times The PVDF membrane was incubated with primary antibodies against Ki-67 (1:1,000; cat. no. ab15580; Abcam), proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. ab280088; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam) and Bax (1:1,000; cat. no. ab182734; Abcam) at 4˚C overnight. Secondary horseradish peroxidase (HRP)-rabbit antibody (1:5,000; cat. no. ab6858; Abcam) was then added to the membrane, after which it was incubated at room temperature for 2 h. The PVDF membrane was then washed with TBST three times and developed using 5 ml enhanced chemiluminescence substrate (Roche Diagnostics; cat. no. 11684817910) for 3 times and ImageJ software (version k 1.45; National Institutes of Health) was used for analysis.
3
Western Blot Analysis of Apoptosis and RNA Modification
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RIPA buffer (Beyotime) was used to extract the total cellular proteins and a BCA assay kit (Beyotime) was used to determine the protein concentration in each extract. A 40 μg sample of protein from each extract was separated by 10% SDS-PAGE and the protein bands were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA), which were subsequently blocked with 5% non-fat milk in TBST for 2 h at room temperature. Next, the membranes were incubated with primary antibodies against caspase-3 (ab32042, Abcam, Cambridge MA, USA), Bax (ab182734, Abcam), Bcl-2 (ab182858, Abcam), PARP1 (ab227244, Abcam), BRCA1 (ab131360, Abcam), BRCA2 (ab239375, Abcam), YTHDF1 (ab220162, Abcam), WTAP (ab232392, Abcam), ALKBH5 (ABE547, Sigma-Aldrich), and β-actin (ab8227, Abcam) at 4 °C overnight, followed by a 1.5 h incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. Finally, the target proteins were visualized with an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).
4
Alisol B Inhibits Kidney Injury through Modulating Inflammatory and Oxidative Pathways
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Alisol B was isolated from A. orientale by authors, and identified through 1H and 13C NMR spectra. Recombinant human sEH was purchased from Cayman Chemical and used to detect the KD value of alisol B with sEH. Hematoxylin and eosin (H&E) staining kit was obtained from Beyotime (Shanghai, China). The primary antibodies were purchased from Proteintech (Wuhan, China), Cell Signaling Technology (CST, Danvers, MA, USA), Abcam (Cambridge, United Kingdom), Boster (Wuhan, China) and Abclonal (Wuhan, China). Detailed information for the primary antibodies were as following: p53 (10442-1-AP, Proteintech), PARP (9542S, CST), Bcl-2 (3498S, CST), Bax (ab182734, Abcam), Caspase3 (ab49822, Abcam), Cleave-Caspase3 (9664S, CST), β-actin (BM0627, Boster), MCP-1 (66272-1-lg, Proteintech), ICAM-1 (60299-1-lg, Proteintech), iNOS (13120S, CST), IL-6 (21865-1-AP, Proteintech), p65 (8242T, CST), Grp78 (ab21685, abcam), 4-HNE (ab48506, Abcam), TNF-α (60291-1-lg, Proteintech), 8-OXO (ab206461, Abcam), HO-1 (A1346, ABclonal), Nrf2 (ab31163, Abcam), Keap1 (10503-2-AP, Proteintech), sEH (10833-1-AP, Proteintech), Kim-1 (14971S, CST), GSK3β (A2081, ABclonal), and p-GSK3β (5558P, CST).
5
Apoptosis-related Protein Analysis
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Western blot analysis was performed to measure the levels of apoptosis-related proteins in parental cells, shCOPB2-treated cells, and shCtrl-treated cells as previously described. Brieflf, total proteins from RKO and HCT116 cells were extracted, separated using 10% SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Bedford, UK). After blocking in 5% skim milk at 37°C for 1 h, the membranes were incubated with various antibodies, including antibodies againse COPB2 (Sigma-Aldrich, HPA036867), BCL-2 (Abcam, ab185002), Bax (Abcam, ab182734), c-Jun (Abcam, ab32137), JNK (Abcam, ab179461), p53 (CST, #2527), Bad (Abcam, ab32445), Caspase3 (Abcam, ab32042) and GAPDH (Abcam, ab181602) overnight at 4°C. Then the membranes were subsequently washed 3 times with TBST and incubated with fluorescently labeled secondary antibodies for 1–2 h at room temperature. Bands were visualized using an Odyssey detection system (Licor Biosciences, Nebraska, USA). The experiment was repeated 3 times.
6
Quantifying Apoptosis Regulators via Western Blot
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After 10% SDS-PAGE separation, extracted total protein was wet-transferred to the PVDF membrane (Sigma-Aldrich, USA). Immunoblots were generated after addition of primary antibodies: anti-bcl-2 antibody (ab182858, Abcam, UK), anti-bax antibody (ab182734, Abcam), anti-CEBPB antibody (ab32358, Abcam), anti-SLC16A3 antibody (ab74109, Abcam), and anti-GAPDH antibody (ab171091, Abcam). Horseradish peroxidase-labeled IgG and enhanced chemiluminescence solution were added for visualization. With GAPDH as the loading control, immunoblots were photographed by the use of Bio-Rad image analysis system (BIO-RAD, Hercules, CA) and their densimetric analyses were performed using Quantity One v4.6.2 software.
7
Western Blot Analysis of Apoptotic Markers
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The radio immunoprecipitation assay (RIPA) (Takara, Dalian, China) was introduced to isolate the protein. The BCA protein detection kit (Junxin, Suzhou, China) was applied to measure the concentration of protein. The proteins (30 μg in each lane) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in 5% fat-free milk and exposed to different primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated IgG. The immunoreactive protein bands were detected with a chemiluminescence (ECL) reagent (Junxin, Suzhou, China) and the Bio-Rad ChemiDocTM XRS system. β-Actin was measured as a loading control. The antibodies used in this study are listed below: The anti-PTEN antibody (Ab267787, Abcam, Cambridge, British), anti-Bcl-2 antibody (Ab32124, Abcam, Cambridge, British), anti-Bax antibody (Ab182734, Abcam, Cambridge, British), anti-β-Actin (Ab207604, Abcam, Cambridge, British) and Goat Anti-Rabbit IgG H&L (HRP) (Ab 6721, Ab207604, Abcam, Cambridge, British) were used according to the protocol.
8
Western Blot Analysis of Apoptosis and PI3K/AKT Signaling
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After cell treatment, the medium was discarded. Supplementation of protein lysis solution (Roche) was completed for separating the total protein. Then, 50 μg of total protein was sampled on 12% polyacrylamide gel for 2-hour electrophoresis at 100 V. Afterward, the electrophoresed total protein was transferred to polyvinylidene fluoride (PVDF) membranes. One hour after being sealed with 5% skimmed milk at RT, the membranes were flushed three times (10 minutes each time) with Tris-buffered saline with Tween-20 (TBST) for overnight incubation at 4°C with the following primary antibodies (Abcam, Cambridge, UK, concentration 1:1000): Anti-Bax antibody (ab182734), Anti-Bcl-2 antibody (ab692), Anti-Cleaved Caspase-3 antibody (ab214430), Anti-p-AKT antibody (ab38449), Anti-AKT antibody (ab8805), Anti-p-PI3K antibody (ab278545), Anti-PI3K antibody (ab140307), and Anti-HIF-1 alpha Antibody (ab179483). Next, the membranes were maintained with the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit IgG (ab6205718) (concentration 1:2500) at RT for one hour after being rinsed with TBST. Following 3 washes with TBST (10 minutes/time), the membranes were exposed with enhanced chemiluminescence (ECL) chromogenic agent (Millipore, Bedford, MA, USA), and a membrane scanner was utilized for imaging [24 (link)].
9
Protein Expression Analysis of TLR4 Signaling Pathway
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Lung samples were homogenized and qualified with a BCA detecting kit (Beyotime, China). Protein lysates of 30 μg were separated by SDS‐PAGE and transferred onto the PVDF membrane (Millipore, USA). After being blocked with 5% (w/v) nonfat milk, the membranes were then incubated with primary antibodies against TLR4 (1:500, ab13556, Abcam), MyD88 (1:1000, ab219413, Abcam), NF‐κB p65 (1:2000, ab32536, Abcam), Bax (1:1000, ab182734, Abcam), Bcl‐2 (1:2000, ab182858, Abcam), and β‐actin (1:5000, D191047, Sangon Biotech, China) at 4°C overnight. Next, the membranes were incubated with HRP‐conjugated goat anti‐rabbit secondary antibody (1:2000, ab7090, Abcam) for 1 h at room temperature. Finally, the optical density of bands was measured via ImageJ software.
10
Protein Expression Analysis in Cells
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The total proteins in each sample were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology), and the amount of protein in each extract was quantified using a BCA kit (Beyotime, China). Next, an equal amount of protein from each extract (30 µg) was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Bio-Rad, USA), which were subsequently blocked with 5% non-fat milk for 2 h at room temperature. The membranes were then incubated with anti-FZD8 (SAB4503133, Sigma-Aldrich,), anti-Bax (ab182734, Abcam, Cambridge, MA, USA), anti-Bcl-2 (ab194583, Abcam), anti-caspase-3 (ab184787, Abcam), anti-Col2A1 (LS-C800256, LifeSpan Biosciences, Seattle, WA, USA), anti-MMP-13 (ab39012, Abcam), anti-β-catenin (ab32572, Abcam), and anti-GAPDH (ab181602, Abcam) antibodies overnight at 4 °C; after which, they were incubated for 2 h at room temperature with an HRP-conjugated secondary antibody. Enhanced chemiluminescence reagent (Forevergen Biosciences, Guangzhou, China) was used to detect the immunostained protein bands, and GAPDH served as a loading control.
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