The protein levels of Occludin, ZO-1, SIRT1, Bax, Bcl-2, NF-κB p65, histone H3 and β-actin were detected by Western blotting. For p65, nuclear extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The primary antibodies were as follows: rabbit anti-Occludin (1:2000, Proteintech Group, Chicago, IL, USA), rabbit anti-ZO-1 (1:500, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-Sirt1 (1:3000, Millipore, Billerica, MA, USA), mouse anti-Bax (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Bcl-2 (1:1000, Santa Cruz Biotechnology), mouse anti-p65 (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-histone H3 (1:1000, Abcam), and mouse anti-β-actin (1:5000, Sigma-Aldrich).
Mouse anti bax
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-Bax is an antibody that specifically recognizes the Bax protein. Bax is a pro-apoptotic member of the Bcl-2 protein family and plays a key role in the mitochondrial apoptosis pathway.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti bcl 2, by Santa Cruz Biotechnology (6 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (6 mentions)
Mouse anti p53, by Santa Cruz Biotechnology (3 mentions)
Rabbit anti bcl 2, by Santa Cruz Biotechnology (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Mouse anti gr, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Bioworld Technology (2 mentions)
Anti cox 4, by Bioworld Technology (2 mentions)
Anti caspase 3, by Bioworld Technology (2 mentions)
Cleaved caspase 3, by Bioworld Technology (2 mentions)
Anti cytochrome c, by Bioworld Technology (2 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence detection kit, by Santa Cruz Biotechnology (2 mentions)
Colorimetric protein assay kit, by Bio-Rad (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Rabbit anti caspase 3, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti histone h3, by Abcam (1 mentions)
15 protocols using mouse anti bax
1
Western Blot Analysis of Tight Junction and Apoptosis Proteins
2
Western Blot Analysis of Protein Targets
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The cell extracts were subjected to SDS-PAGE and electroblotted onto nitrocellulose membranes. Then, the membranes were blocked with Tris-buffered saline and Tween -20 containing 5% of bovine serum albumin at room temperature for 2 h [49 (link)], following incubation with corresponding antibodies overnight at 4 °C. The membranes were washed and incubated with appropriate dilution of secondary antibodies for 1 h at room temperature. After washing with Tris-buffered saline, the bands were detected under a Tanon 5500 chemiluminescence image system (Tanon Inc., Shanghai, China).
Rabbit anti-MDA5 and mouse anti-Bax antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Rabbit anti-ERK1/2, phospho-ERK1/2, rabbit anti-caspase-3, rabbit anti-caspase-8, rabbit anti-NF-κB2, phosphor-NF-κB2, and rabbit anti-GAPDH antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Rabbit anti-Flag was purchased from Bioss Antibodies (Beijing, China). The HRP conjugated goat anti-rabbit and goat anti-mouse were purchased from MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China).
Rabbit anti-MDA5 and mouse anti-Bax antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Rabbit anti-ERK1/2, phospho-ERK1/2, rabbit anti-caspase-3, rabbit anti-caspase-8, rabbit anti-NF-κB2, phosphor-NF-κB2, and rabbit anti-GAPDH antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Rabbit anti-Flag was purchased from Bioss Antibodies (Beijing, China). The HRP conjugated goat anti-rabbit and goat anti-mouse were purchased from MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China).
3
Western Blot Analysis of Cell Signaling
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Firstly, the cells were washed twice with PBS prior to sample preparation. RIPA lysis solution (P0013C, Beyotime, Shanghai, China) was used to lyse the cells at 4 °C for 30 min. A BCA kit (P0010S, Beyotime, Shanghai, China) was used to detect the total protein concentration. Then, SDS–PAGE loading buffer (P0015F, Beyotime, Shanghai, China) was added to the lysate, and the mixture was boiled at 100 °C for 5 min. Then, 10–20 µg of protein per sample was separated with SDS-PAGE gels. Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA, ISEQ00010). The blots were incubated overnight at 4 °C with the corresponding primary antibodies and incubated with secondary antibodies at room temperature for 1 h, and developed with ECL substrate solutions A and B (Pierce™, Thermo Fischer). The primary antibodies used were: mouse anti-CDK4(sc-23896, Santa Cruz), mouse anti-Cyclin D1(sc-8396, Santa Cruz), mouse anti-p53(sc-126, Santa Cruz), mouse anti-BCL-2(sc-7382, Santa Cruz), mouse anti-BAX (sc-7480, Santa Cruz), mouse anti-NF-kB (sc-8008, Santa Cruz), mouse anti-Caspase3(sc-7272, Santa Cruz), and mouse anti-β-actin (sc-47778, Santa Cruz).
4
Hippocampal Protein Expression Analysis
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Equal amounts of total protein samples from the hippocampus were separated via SDS/PAGE (10% Bis‐Tris gel; Invitrogen), following which they were transferred onto a PVDF membrane (Bio‐Rad Laboratories) and blocked in TBST buffer (25 mM Tris‐HCl, 160 mM sodium chloride, and 0.05% Tween‐20) containing 5% (wt/vol) bovine serum albumin (Santa Cruz Biotechnology) for 1 hr at 25°C. The membrane was then incubated with primary antibodies overnight at 4°C. This was followed by incubation with the corresponding secondary antibody for 1 hr at room temperature. Primary antibodies included mouse anti–cleaved caspase‐3 (1:400, Cell Signaling Technology), mouse anti‐Bax (1:1000, Santa Cruz Biotechnology), mouse anti‐Bcl‐2 (1:500, Santa Cruz Biotechnology), mouse anti‐p65 (1:500, Santa Cruz Biotechnology), and rabbit anti‐GAPDH (1:600, Cell signaling Technology). Immunoblot bands were visualized using an ECL Western blot kit (NeoBioscience Biotechnology, China). An Eastman Kodak Image Analyzer (Rochester, NY) was used for semiquantification of the protein signal intensity.
5
Hippocampal Bax and Bcl-2 Expression Analysis
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Bax and Bcl-2 expressions were determined by Western blot analysis, according to the previously described method (Kim et al., 2010 (link)). The hippocampal tissues were dissected and collected, and then were immediately frozen at −70°C. The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50-mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) (pH, 7.5), 150-mM NaCl, 10% glycerol, 1% Triton X-100, 1-mM PMSF (phenylmethylsulfonyl fluoride), 1-mM EGTA (ethyleneglycol-bis-(b-aminoethylether)-N,N,N′, N′-tetraacetic acid), 1.5-mM MgCl2·6H2O, 1-mM sodium orthovanadate, and 100-mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membranes were incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated overnight at 4°C with the following primary antibodies: mouse anti-β-actin, mouse anti-Bcl-2, and mouse anti-Bax (1:1,000; Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 hr with attempt secondary antibodies (1:2,000; Vector Laboratories), and band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).
6
Molecular Analyses of Skeletal Muscle
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Protein lysates were extracted from frozen tibialis anterior (TA) muscles using RIPA lysis buffer (Millipore) containing protease and phosphatase inhibitor cocktail (Roche Diagnostics). Aliquots of 20 μg lysates were resolved on a NuPage 4–12% SDS-PAGE. Western blotting was carried out using the following primary antibodies: rabbit anti-LC3, anti-p62 (Cell signaling technology), anti-BCL-2 (Abcam), anti-HSPH1 (Sigma Aldrich), anti-HSPA4L, anti-HSPA4, mouse anti-BAX and anti-GAPDH (Santa Cruz Biotechnology). For quantification, an enhanced chemiluminescence detection system (Amersham Bioscience) and Image Lab software (Bio-Rad) were used according to the manufacturer’s instructions.
7
Immunoblotting Analysis of Cell Signaling Proteins
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Protein was extracted in RIPA lysis buffer and identified by BCA assay. We separated denatured protein in SDS-polyacrylamide gel and transferred it to the Hybond membrane, which was blocked with 5% milk in TBST. For immunoblotting, the membrane was incubated for 1 h with mouse anti-Bax (Santa Cruz), rabbit anti-cdc25B (H-85, Santa Cruz), anti-cdc25C (C-20, Santa Cruz), anti-E-cadherin (Cell Signaling Technology), anti-N-cadherin (Cell Signaling Technology), anti-slug (Cell Signaling Technology), anti-twist1 (Cell Signaling Technology), or anti-GAPDH (Santa Cruz) antibody for 1 h in TBST at room temperature. Subsequently, these membranes were incubated with antimouse or antirabbit IgG conjugated to horseradish peroxidase (Dako) for 1 h at room temperature. Bands were visualized using ECL- Plus (Santa Cruz).
8
Subcellular Protein Fractionation and Western Blot
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Nuclear and cytoplasmic proteins were separately fractionated from the harvested cells using NE-PER ® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) as previously described [15] . The primary antibodies used were rabbit anti-Nrf2 (abcam, Cambridge, UK), rabbit anti-HO-1 (abcam), rabbit anti-LC3 (NOVUS Biologicals, Littleton, CO, USA), mouse anti-cytochrome C, mouse anti-Bcl-2, mouse anti-Bax, mouse anti-Lamin B1, and mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies used were anti-rabbit or anti-mouse immunoglobulin G, conjugated to horseradish peroxidase (Santa Cruz Biotechnology).
9
Apoptosis Regulators in RPE Cells
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RPE cells were treated with 1 μM A-1155463, 1 μM 4NQO or both compounds. Control cells were treated with 0,1% DMSO. After 1 and 2 h cells were fixed using 3.3% formaldehyde in 10% FBS and PBS, pH 7.4. Cells were permeabilized using 10% FBS and 0.1% Triton X-100 in PBS. Bad, Bax (2 different antibodies), Bcl-xL, p53 and Tom40 were detected using rabbit anti-Bcl-xL (1 : 250; clone 54H6; Cell Signalling Technology), mouse anti-Bax (1:250; 2D2, sc-20067), rabbit anti-Bax (1:250; N-20, sc-493), rabbit anti-BAD (1:250; clone D24A9; Santa Cruz Biotechnology), mouse anti-p53
(1 : 250; Santa Cruz Biotechnology) and anti-Tom40 (1:250; Santa Cruz Biotechnology, sc-11414) antibodies were used. Secondary goat anti-mouse IgG (H+L) Alexa Fluor ® 488 and 568 (Thermo Scientific), goat anti-rabbit IgG (H+L) Alexa Fluor ® 488 and 568 (Thermo Scientific) antibodies were used. ProLong™ Gold Antifade Mountant (Invitrogen) with DAPI was used for mounting of cells. Cells were imaged using Zeiss LSM710 confocal microscope.
(1 : 250; Santa Cruz Biotechnology) and anti-Tom40 (1:250; Santa Cruz Biotechnology, sc-11414) antibodies were used. Secondary goat anti-mouse IgG (H+L) Alexa Fluor ® 488 and 568 (Thermo Scientific), goat anti-rabbit IgG (H+L) Alexa Fluor ® 488 and 568 (Thermo Scientific) antibodies were used. ProLong™ Gold Antifade Mountant (Invitrogen) with DAPI was used for mounting of cells. Cells were imaged using Zeiss LSM710 confocal microscope.
10
Hippocampal Protein Profiling
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Extracted the cytoplasm and mitochondria protein from the hippocampus, and quantified the protein concentrations with BCA (Beyotime, China). Loading buffer (0.1 MTris-HCl buffer (pH 6.8) containing 0.2 M DTT, 4% SDS, 20% glycerol and 0.1% bromophenol blue) was used to dissolve 40-60 µg equal volume protein samples. The samples were separated on 10% SDS-PAGE and then electrically transferred to PVDF membrane at 90 V. PVDF membranes were incubated with TBST (containing 5% skimmed milk) for 1 h at 37 °C and with primary antibodies at 4 °C for 24 h. The primary antibodies used were as follows: mouse anti-Bax, mouse anti-GR (1:1000), mouse anti-bcl-2, (1:400, SantaCruz Biotechnology, USA), antiβ-actin, anti-Cox-IV, anti-caspase-3, cleaved caspase-3, anti-cytochrome c (1:1000, Bioworld Technology, China). The blots were thoroughly washed with TBST and incubated at 37 °C with the secondary antibody in TBST containing 5% skimmed milk powder for 1 h. After that, the signal was tested by enhanced chemiluminescence (ECL kit, Millipore, USA). Cox-IV was used as an internal reference for proteins in mitochondria, while β-actin was used in the cytoplasm. The membranes were imaged and analyzed using the Quantity One Image Analysis Software (Syngene, U.K.) (49) .
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