Cells were lysed, and the total protein was obtained using RIPA buffer (Beyotime Institute of Biotechnology). A bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to quantify the total protein. Equal amounts of protein were separated by 12% SDS-PAGE for 40 min and then transferred to PVDF membranes (EMD Millipore). The membranes were blocked for 1.5 h at room temperature with 5% nonfat milk and incubated with primary antibodies including anti-FA2H (Cat no. ab128917; 1:1,000; Abcam), anticleaved caspase-3 (Cat no. ab32042; 1:1,000; Abcam), and anti-pro-caspase-3 (Cat no. ab32499; 1:1,000; Abcam) overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated antirabbit secondary antibody (Cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 2 h. Protein bands were visualized by enhanced chemiluminescence (GE Healthcare Life Sciences). β-Actin (1:1,000; Abcam) served as the loading control for normalization.
Anti pro caspase 3
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-pro-caspase-3 is a primary antibody that binds to the pro-caspase-3 protein. Pro-caspase-3 is an inactive precursor of the caspase-3 enzyme, which plays a critical role in the execution phase of cell apoptosis (programmed cell death). This antibody can be used to detect the pro-caspase-3 protein in various applications, such as western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Abcam (7 mentions)
Anti gapdh, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti bax, by Abcam (3 mentions)
Anti akt, by Abcam (3 mentions)
Anti p akt, by Abcam (3 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti pi3k, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg secondary antibody, by Cell Signaling Technology (2 mentions)
Anti active caspase 3, by Abcam (2 mentions)
Anti mmp2, by Abcam (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
β actin, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Abcam (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
14 protocols using anti pro caspase 3
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from cell lysis. The expressions of caspase 3 (anti-pro-caspase 3 from Abcam plc.; anti-cleaved-caspase 3 from Cell Signaling Technology, Inc.) and caspase 9 (anti-caspase 9, from Abcam plc.) in cell lysis were analyzed using 12% SDS-PAGE, whereas DAPK1 (anti-DAPK1, from Sigma-Aldrich Co., St Louis, MO, USA.), AKT (anti-AKT, from Cell Signaling Technology, Inc.) and phosphorylated AKT (anti-phos-AKTser473, from Cell Signaling Technology, Inc.) were analyzed using 10% SDS-PAGE. β-Actin (anti-β-actin, from #TA-09, ZSGB-BIO, China) was the reference control. After being resolved by SDS-PAGE, proteins were transferred to Polyvinylidene Fluoride (PVDF) membranes and then blocked with 5% bovine serum albumin (Sigma-Aldrich Co.) for 1 h. Next, the membranes were separately incubated with primary antibodies at 4°C overnight, and then with appropriate HRP-conjugated secondary antibody (from #ZDR-5306 and -5307, ZSGB-BIO) at room temperature for 1 h. After detecting signals using ECL reagents (Merck Millipore, Billerica, MA, USA), gray value of different proteins was quantified with ImageJ v1.28 and normalized to β-actin.
3
Western Blot Analysis of Signaling Proteins
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Cells were lysed using RIPA lysis buffer (Beyotime, China), and the protein concentrations were measured using the Bicinchoninic Acid Protein Assay Kit (Beyotime, China). The proteins were separated on a 10% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany), as described previously28 (link). Following 2 h of blocking with TBST supplemented with 5% non-fat dried milk, the membranes were incubated overnight with specific primary antibodies at 4 °C. Anti-PI3K (1:1000, Abcam), anti-Bcl-2 (1:1000, Abcam), anti-AKT (1:2000, Abcam), anti-pAKT (1:500, Abcam), anti-BAX (1:1000, Abcam), anti-LC3B-I (1:500, Abcam), anti-p62 (1:2000, Abcam), anti-mTOR (1:1000, Abcam), anti-cleaved caspase 3 (1:1000, Abcam), and anti-pro-caspase 3 (1:1000, Abcam) were used as primary antibodies. A next step involved washing the membranes, exposing them to peroxidase-conjugated secondary antibodies, and developing them with ELC (Amersham Pharmacia, UK); incubation at room temperature for another 60 min was performed with goat anti-rabbit IgG (1:5000) or goat anti-mouse IgG (1:5,000). Thermo provided the software and hardware for measuring immunoreactivity. The Super Signal West Femto Maximum Sensitivity Substrate Kit was used on a C-Digit Blot Scanner to measure immunoreactivity.
4
Protein Expression Profiling in Min6 Cells
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Total protein was collected from Min6 cells (5×104 cells per well) lysed with RIPA lysis buffer (Vazyme Beyotime), and protein concentrations determined using BCA™ Protein Assay Kits (Solarbio; Beijing, China). Aliquots of 40 μg of protein were loaded onto each lane, and the proteins separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with 5% skim milk and immunoblotted with primary anti-p-PI3K, anti-PI3K, anti-p-AKT, anti-AKT, anti-cleaved caspase-3, anti-pro caspase-3 and anti-GAPDH (Abcam, Cambridge, UK) antibodies overnight at 4°C, following by incubation with the respective secondary antibodies (Abcam) for 1 h at room temperature. The protein bands were visualized using ECL Kits (BestBio, Shanghai, China) and quantified with Image J Software (Bio-Rad, Shanghai, China). Each experiment was performed three times, all on cells of the same passage number.
5
Xanthohumol Cytotoxicity and Apoptosis in Cancer Cells
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XN (Figure 1 ) was
provided by Yumen Tuopu Scientific and Technological Development Co., Ltd.,
purity > 98% (Yumen, Gansu, China). The cell culture medium RPMI-1640 was
purchased from Gibco Laboratories (Grand Island, NY, USA). Neonatal bovine serum
was obtained from Lanzhou Minhai Bio-engineering Co., Ltd. (Lanzhou, China).
Streptomycin, penicillin, L-glutamine, and dimethyl sulfoxide (DMSO) were all
greater than 98% in purity (Sigma-Aldirch Corp.). The
3-(4,5-dimethylthiazo-2-yl) −2,5 -diphenyl-tetrazolium bromide (MTT) kit and
Annexin V-FITC/PI apoptosis detection kit were obtained from KeyGEN Biotech
(Nanjing, China). RIPA Lysis Buffer and Enhanced BCA Protein Assay Kit were
purchased from Beyotime (Shanghai, China). Collagenase type II was purchased
from Worthington Biochemical Corp. (Lakewood, NJ, USA). Williams’ medium E was
obtained from Sigma-Aldrich Japan (Tokyo, Japan). Anti- Pro-caspase-3、Anti-
PRAP-1、Anti-β-actin receptor rabbit monoclonal antibody and rabbit IgG
monoclonal isotype control were purchased from Abcam (Cambridge, MA, USA). All
other chemicals made in China were analytical grade.![]()
provided by Yumen Tuopu Scientific and Technological Development Co., Ltd.,
purity > 98% (Yumen, Gansu, China). The cell culture medium RPMI-1640 was
purchased from Gibco Laboratories (Grand Island, NY, USA). Neonatal bovine serum
was obtained from Lanzhou Minhai Bio-engineering Co., Ltd. (Lanzhou, China).
Streptomycin, penicillin, L-glutamine, and dimethyl sulfoxide (DMSO) were all
greater than 98% in purity (Sigma-Aldirch Corp.). The
3-(4,5-dimethylthiazo-2-yl) −2,5 -diphenyl-tetrazolium bromide (MTT) kit and
Annexin V-FITC/PI apoptosis detection kit were obtained from KeyGEN Biotech
(Nanjing, China). RIPA Lysis Buffer and Enhanced BCA Protein Assay Kit were
purchased from Beyotime (Shanghai, China). Collagenase type II was purchased
from Worthington Biochemical Corp. (Lakewood, NJ, USA). Williams’ medium E was
obtained from Sigma-Aldrich Japan (Tokyo, Japan). Anti- Pro-caspase-3、Anti-
PRAP-1、Anti-β-actin receptor rabbit monoclonal antibody and rabbit IgG
monoclonal isotype control were purchased from Abcam (Cambridge, MA, USA). All
other chemicals made in China were analytical grade.
Chemical structure of Xanthohumol.
6
Western Blot Analysis of Cleaved Caspase-3
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Total protein was extracted from cells using RIPA buffer (Beyotime Institute of Biotechnology) and quantified using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (40 µg per lane) were separated via 12% SDS-PAGE for 40 min and transferred to PVDF membranes (EMD Millipore). The membranes were blocked for 1.5 h at room temperature with 5% non-fat milk. Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-cleaved caspase-3 (cat no. ab32042; 1:1,000; Abcam), anti-pro-caspase-3 (cat no. ab32499; 1:1,000; Abcam) and GAPDH (cat no. ab9485; 1:1,000; Abcam). Following primary incubation, the membranes were incubated with an anti-rabbit horseradish peroxidase conjugated IgG secondary antibody (cat no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 2 h. Protein bands were visualized using the enhanced chemiluminescence method (Cytiva). GAPDH was used as the loading control.
7
Apoptosis Pathway Protein Analysis
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Protein was extracted via RIPA lysis buffers (Beyotime, China), which was assessed with a bicinchoninic acid protein assay kit (Beyotime). The sample was separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. Then, the membrane was blocked using 5% non-fat milk for 1 h, which was incubated overnight with primary antibodies at 4°C and secondary antibody (1:2,000; Abcam, USA) lasting 1 h at room temperature. The primary antibodies included anti-Bax (1:1,000; Abcam), anti-Bcl2 (1:1,000; Abcam), anti-cleaved-caspase-3 (1:1,000; Abcam), anti-pro-caspase-3 (1:1,000; Abcam), anti-cleaved-caspase-8 (1:1,000; Abcam), anti-pro-caspase-8 (1:1,000; Abcam), anti-cleaved-caspase-9 (1:1,000; Abcam), anti-pro-caspase-9 (1:1,000; Abcam), and anti-GAPDH (1:1,000; Abcam). GAPDH served as a control. Image Lab™ Software (Bio-Rad, China) was used to quantify the intensity of blots.
8
Western Blot Quantification of Apoptosis Markers
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Tissue proteins were obtained using RIPA lysis buffer (Biosharp). Protein concentrations were quantified by the Pierce Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific). The proteins were transferred to the polyvinylidene fluoride membrane after being separated with a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and sealed in 5% skim milk and then incubated with primary antibody at 4°C overnight. All the primary antibodies we used were as follows: anti-APAF1 (1:2000; Abcam, Cambridge, UK), anti-clv-caspase-3 (1:2000; Abcam), anti-pro-caspase-3 (1:2000; Abcam), anti-N-GSDME (1:2000; Abcam), and anti- GAPDH (1:1000, Abcam). The membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:5000; Abcam) for 1 h. The bands were examined using Tanon 5200 Automatic chemiluminescence image analysis system (Shanghai, China). Enhanced chemiluminescence solution was applied for color development to observe images.
9
Western Blot Analysis of Cell Signaling Pathways
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Total protein was isolated from cell lysates with radio-immunoprecipitation assay buffer and quantified with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The proteins were resolved on 10% SDS and subsequently transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). Following blocking, the membranes were incubated with primary antibodies at 4°C overnight and subsequently incubated with an anti-rabbit secondary antibody (Abcam; 1:5,000) at room temperature for 1 h. The membranes were scanned on an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences). The primary antibodies used in the present study were as follows: anti-CDKN1B (Abcam; 1:1,000), anti-SATB2 (Abcam; 1:1,000), anti-ATF4 (Abcam; 1:1,000), anti-Runx3 (Abcam; 1:1,000), anti-Cyclin E1 (Abcam; 1:1,000), anti-CDK2 (Abcam; 1:1,000), anti-Bax (Abcam; 1:1,000), anti-X-linked inhibitor of apoptosis protein (XIAP, Abcam; 1:1,000), anti-pro-caspase 3 (Abcam; 1:1,000), anti-active caspase 3 (Abcam; 1:1,000) and anti-β-actin (Abcam; 1:1,000). β-actin was used as an internal control.
10
Protein Expression Analysis by Western Blot
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An equal amount of proteins from whole-cell lysates were separated by 4–12% SDS-PAGE and were transferred to a nitrocellulose membrane. Blots were blocked for 1 h with 5% non-fat dry milk and then incubated over night with the following primary antibodies: anti-CA-IX (R&D), anti-N-cadherin, anti-β-catenin and anti-Vimentin (CST-9782), anti-pro-Caspase-3, anti-Cleaved-Caspase-3, anti-AKT (CST-9272), anti-pAKT (CST-9271), anti-ERK (CST-9102), anti-pERK (CST-9101), anti-MMP-2 (CST-33437), anti-CD44 (Abcam), anti-GADPH (G8795) and anti-Actin (A4700). After washing with 0.1% Tween-20 in PBS, the filters were incubated with their respective secondary antibodies for 1 h and analyzed using the ECL system. Densitometric analyses were performed on at least two different expositions to assure the linearity of each acquisition using ImageJ software (v1.46r) [23 (link)].
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