Total proteins were extracted through a Total Protein Extraction Kit (KeyGen Biotech, Nanjing, China). Concentrations of protein were examined under BCA commercial kit (KeyGen). Proteins were separated using SDS-PAGE and then moved onto PVDF membranes (Millipore, Carlsbad, USA). Membranes were sealed in non-fat milk and then incubated with given primary antibodies: anti-MMP2 (1:1000, ab37150, Abcam, Cambridge, USA), anti-MMP7 (1:1000, ab5706, Abcam), anti-Bcl-2 (1:1000, ab32124, Abcam), anti-bax (1:1000, ab32503, Abcam), anti-caspase 3 (1:1000, ab13847, Abcam), anti-cleaved caspase-3 (1:1000, ab2302, Abcam), anti-caspase 8 (1:1000, ab25901, Abcam), anti-cleaved caspase-8 (1:1000, ab25901, Abcam), anti-caspase 9 (1:1000, ab32539, Abcam), anti-cleaved caspase-9 (1:1000, ab2324, Abcam), anti-E-cadherin (1:1000, ab15148, Abcam), anti-N-cadherin (1:1000, ab76057, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam). GAPDH was used as a measurement control for other proteins. Moreover, the membranes were co-cultured with goat anti-mouse IgG H&L (Cy3®) preadsorbed (1:2000, ab97035, Abcam) secondary antibodies for 1 h darkness. Chemiluminescence system (Invitrogen) was employed to observe the protein bands.
Ab25901
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab25901 is a laboratory product manufactured by Abcam. It is a type of lab equipment, but a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab13847, by Abcam (5 mentions)
Ab32503, by Abcam (5 mentions)
Ab32124, by Abcam (4 mentions)
Ab6721, by Abcam (3 mentions)
Ab32539, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Ab52298, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab15592, by Proteintech (2 mentions)
Ab8245, by GeneTex (2 mentions)
Chemiluminescence system, by Cytiva (2 mentions)
Gtx30203, by Abcam (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab137550, by Abcam (2 mentions)
Bca kit, by Beyotime (2 mentions)
Pvdf microporous membrane, by Merck Group (2 mentions)
Ab4052, by Abcam (2 mentions)
Ab62557, by Abcam (2 mentions)
26 protocols using ab25901
1
Western Blot Analysis of Protein Markers
2
Western Blot Analysis of Apoptosis Markers
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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.
3
Hippocampus Protein Extraction and Analysis
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Protein was extracted from the hippocampus tissues according to standard Invent protocols (Invent, China) as in our previous study (Li et al., 2020 (link)). The total, nuclear, and total member proteins from the hippocampus tissues were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), blotted and probed with the following antibodies: anti-IFITM3 (Abcam, ab15592, 1:1,000), anti-TLR4 (ProteinTech, 10,745-1-AP, 1:1,000), anti-NF-κB (ProteinTech, 66,350–1-Ig, 1:1,000), anti-β-actin (ProteinTech, 6,008–1-Ig, 1:2,000), anti-GAPDH (Abcam, ab8245, 1:2,000), anti-Na/K-ATPase (GeneTex, GTX30203), anti-cleaved caspase-8 (Abcam, ab25901, 1:1,000), and anti-cleaved caspase-3 (Abcam, ab13847, 1:1,000). Bradford assays (Bio-Rad Laboratories, Hercules, CA, United States) were used to quantify the protein concentrations. The blots were visualized with a chemiluminescence system (Amersham Bioscience, Buckinghamshire, United Kingdom), and the signals were quantified by densitometry. ImageJ was used to measured densities of blots, and the relative density was the ratio of target protein to GAPDH.
4
Western Blot Analysis of Apoptosis Signaling
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Cells were lysed on ice, then 30 µg proteins were separated on 10% SDS-PAGE gels and transferred onto a polyvinyl difluoride (PVDF) membrane. After blocked with 5% skimmed milk, the membrane was incubated with one of the following antibodies overnight at 4 °C: anti-ASK-1 antibody (8662, Cell Signaling Technology), anti-phospho-ASK-1 antibody (3764, Cell Signaling Technology), anti-STRAP antibody (AP29336; One World Lab), anti-14-3-3 antibody (9636; Cell Signaling Technology), anti-caspase 8 antibody (ab25901, Abcam), anti-TNF-α antibody (ab1793, Abcam), and anti-tubulin antibody (ab6160, Abcam). Then the membrane was incubated with secondary antibody (ab6721 or ab6789, Abcam) for 1 h at room temperature. Finally, the membrane was photographed by ECL Imaging System (Tanon 6060, USA).
5
Immunofluorescence Analysis of Autophagy and Apoptosis Markers
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Twenty-four hours after transfection, the ACHN cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.01% Triton X-100 for 20 min at room temperature. After that, we had them blocked with 5% goat serum for 1 h at 37°C and performed an incubation with primary antibodies overnight at 4°C. The secondary antibodies were Alexa 488-conjugated goat anti-mouse or anti-rabbit IgG (Abcam, UK). Implementation of 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) was for counterstaining the nuclei. Then, we took out fluorescent images with fluorescence microscope (IX70, Olympus, Japan). The primary antibodies were purchased from Abcam as follows: anti-LC3B (no. ab225383, 1 : 1000), anti-TRAF6 (no. ab40675, 1 : 500), anti-TP53INP2 (no. ab273012, 1 : 100), and caspase-8 (no. ab25901, 1 : 100).
6
Apoptosis Pathway Analysis Protocol
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Dulbecco’s Modified Eagle Medium—high glucose (DMEM-HG) (12800-58), Roswell Park Memorial Institute (RPMI)-1640, DMEM/Ham’s F-12 (GIBCO-Invitrogen, Carlsbad, CA, USA), fetal bovine serum (FBS), phosphate-buffered saline (PBS)and trypsin-EDTA solution were purchased from Gibco (Grand Island, NY, USA) Dimethyl sulfoxide (DMSO), and sulforhodamine B (SRB) was purchased from Sigma Chemical, Inc. (St Louis, MO, USA) The substrate of caspase-9 (LEHD-para-nitroaniline; LEHD-p-NA), caspase-8 (IETD-para-nitroaniline; IETD-p-NA), caspase-3 (DEVD-para-nitroaniline; DEVD-p-NA), and SuperSignal West Pico Chemiluminescent Substrate were obtained from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA, USA). Primary antibodies against caspase-9 (ab32539), caspase-8 (ab25901), caspase-7 (ab25900), BAX (ab32503), Bcl-xl (ab32370), Bid (ab2388), Noxa (ab13654), actin (ab8227) and peroxidase-labeled secondary antibodies; anti-rabbit IgG (ab97051), anti-mouse IgG (ab97046) were purchased from Abcam (Cambridge, UK). Protease inhibitor was obtained from Roche Diagnostics, Mannheim, Germany.
7
Apoptosis Signaling Pathway Analysis
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MCF-7 and BT474 cells were harvested by scraping and lysed in RIPA buffer followed homogenized at 4°C for 10 min. Protein were analyzed by SDS-PAGE assays followed transfer membrane. Protein were incubated with rabbit anti-human Bcl-2 (1:400, ab32124), Bcl-w (1:500, ab2568), caspase-3 (1:500, ab217), caspase-8 (1:400, ab25901), PI3K (1:400, ab86714), AKT (1:400, ab8805), mTOR (1:400, ab2732), and β-actin (1:400, ab5694) (Abcam, Shanghai, China) for 12 h at 4°C. The HRP-labeled secondary goat anti-rabbit antibodies (1:5,000; Abcam) were incubated and performed to analysis the proteins expression by using using chemiluminescence detection system.
8
Immunohistochemical Analysis of Oxidative Stress Markers
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After dewaxing and hydration, the sections were placed in citrate buffer and heated to 95°C for 20 min. After the sections cooled naturally, we treated the sections with 3% H2O2 for half an hour. After washing the sections with phosphate-buffered saline (PBS), we treated the sections with 10% goat serum for 1 h. We then treated the sections with SOD1 (ab13498, Abcam, Cambridge, MA, USA), SOD2 (ab13534, Abcam, Cambridge, MA, USA), NF-E2-realted factor2 (Nrf2) (ab137550, Abcam, Cambridge, MA, USA), caspase8 (ab25901, Abcam, Cambridge, MA, USA), and caspase9 (ab32539, Abcam, Cambridge, MA, USA) antibodies at 4°C overnight. The next day, we treated the sections with the universal secondary antibody from the IHC staining kit (GeneTech, Shanghai, China) for 1 h. Finally, we used the developer in the IHC staining kit for color development.
9
Hippocampus Protein Extraction and Analysis
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Protein was extracted from the hippocampus tissues according to standard Invent protocols (Invent, China) as in our previous study (Li et al., 2020 (link)). The total, nuclear, and total member proteins from the hippocampus tissues were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), blotted and probed with the following antibodies: anti-IFITM3 (Abcam, ab15592, 1:1,000), anti-TLR4 (ProteinTech, 10,745-1-AP, 1:1,000), anti-NF-κB (ProteinTech, 66,350–1-Ig, 1:1,000), anti-β-actin (ProteinTech, 6,008–1-Ig, 1:2,000), anti-GAPDH (Abcam, ab8245, 1:2,000), anti-Na/K-ATPase (GeneTex, GTX30203), anti-cleaved caspase-8 (Abcam, ab25901, 1:1,000), and anti-cleaved caspase-3 (Abcam, ab13847, 1:1,000). Bradford assays (Bio-Rad Laboratories, Hercules, CA, United States) were used to quantify the protein concentrations. The blots were visualized with a chemiluminescence system (Amersham Bioscience, Buckinghamshire, United Kingdom), and the signals were quantified by densitometry. ImageJ was used to measured densities of blots, and the relative density was the ratio of target protein to GAPDH.
10
Western Blot Analysis of Apoptotic Markers
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Western blot analysis was performed on renal lysates or cell extracts using the following primary antibodies: anti-DcR2 (ab108421; Abcam), anti-FLIP (ab8421; Abcam), anti-cleaved caspase 3 (ab214430; Abcam), anti-caspase 8 (ab25901; Abcam), caspase 3 (ab184787; Abcam), anti-caspase 7 (ab255818; Abcam), anti-cleaved caspase 7 (ab256469; Abcam), Akt (sc5298, Santa Cruz), pAkt (sc135650, Santa Cruz), and anti-GAPDH (BM3876; Boster). The intensity of each band was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).
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