Immunoblotting was performed as described previously21 (link). The anti-KAP3, anti-laminin, and anti-β-actin antibodies were used for immunoblotting at dilutions of 1:500, 1:400, and 1:2000, respectively. For immunodetection, anti-rabbit IgG or anti-mouse IgG antibody (Cell Signaling Technology, Beverly, MA, USA) was used as the secondary antibody at a dilution of 1:5000 or 1:10,000, respectively. Antibody binding was detected using the Enhanced ChemiLuminescence (ECL) system (GE Healthcare, Chicago, IL, USA).
Anti mouse igg antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-mouse IgG antibody is a laboratory reagent used to detect and quantify mouse immunoglobulin G (IgG) in various experimental applications. It binds specifically to the Fc region of mouse IgG molecules, allowing for their identification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti rabbit igg conformation specific l27a9, by Cell Signaling Technology (4 mentions)
Ecl prime western detection kit, by GE Healthcare (4 mentions)
Protein a g conjugated agarose beads, by Santa Cruz Biotechnology (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Anti rabbit igg antibody, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (3 mentions)
Hrp conjugated anti rabbit igg antibody, by Cell Signaling Technology (2 mentions)
Monoclonal antibody to tab1 or p38, by Cell Signaling Technology (2 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti myc tag, by Cell Signaling Technology (2 mentions)
Anti caspase 9, by Enzo Life Sciences (2 mentions)
Anti k 63 ub, by Abcam (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Anti hsp90, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
30 protocols using anti mouse igg antibody
1
Immunoblotting of KAP3, Laminin, and β-Actin
2
Western Blot Analysis of Proteins
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Total cell lysates were prepared in RIPA lysis buffer (Beyotime Biotechnology) supplemented with 1 mM PMSF and 1× protease inhibitor cocktail (Yeasen), and nuclear protein was extracted by nuclear protein extraction kit (Solarbio) in accordance with the manufacturer’s instructions. The samples were separated by SDS-PAGE and transferred to the PVDF membrane (Millipore). The membranes were incubated with the indicated primary antibodies at 4 °C overnight and then incubated with an HRP-conjugated anti-rabbit IgG antibody (Cell Signaling, Cat# 7074, 1:5000) or anti-mouse IgG antibody (Cell Signaling, Cat# 7076, 1:5000) at room temperature for 1 h. The signal was visualized by using the NcmECL Ultra kit (New Cell & Molecular Biotech) and then was exposed by using MiniChemi (SageCreation).
3
Immunoblot Analysis of Phosphorylated RyR2
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Immunoblot analysis was performed as previously described36 . Heart tissue was homogenized in ice-cold RIPA buffer containing protease inhibitor (cOmplete Protease Inhibitor Cocktail, Merck)) and phosphatase inhibitor (PhosSTOP, Sigma). Supernatant was collected after centrifugation at 15,000 × g and an aliquot containing equal amount of protein was mixed with loading buffer (125 mM Tris-HCl pH 6.8, 30% glycerol, 10% SDS, and 0.6 M DTT). Without heat denaturation, protein sample was subjected to SDS–PAGE and transferred to PVDF membrane (Thermo Fisher Scientific). Specific antibodies against phospho-RyR2(Ser2808) (1:1000 dilution), phospho-RyR2(Ser2814) (1:1000 dilution; Badrilla)36 –39 (link), β-actin (1:2500 dilution; Invitrogen)40 (link)–42 (link) were used as primary antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:3000 dilution; Cell Signaling Technology) and anti-mouse IgG antibody (1:3000 dilution; Cell Signaling Technology) were used as secondary antibodies. Immunoreactive signals were detected with ECL prime Western Blotting Detection System (GE Healthcare).
4
Co-immunoprecipitation Analysis of Protein Interactions
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For co-immunoprecipitation analysis, cell-lysates were prepared using ice-cold HNGT buffer (50 mM HEPES, pH 7.5, 150 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Lysates from 20 × 106 cells were used for immunoprecipitation analysis. For the minor CD4+ CD28− CD27− subset, cells from two separate individuals were pooled to obtain sufficient material for the assay. Extracts were incubated with the indicated antibodies at 4 °C on a rotary shaker overnight, followed by incubation with protein A–G conjugated agarose beads (Santa Cruz Biotechnology) at 4 °C for 3 h. Samples were washed and analyzed by immunoblotting as indicated. Co-immunoprecipitated proteins were detected using Mouse Anti-rabbit IgG Conformation Specific (L27A9; Cell Signalling) or Mouse Anti-rabbit IgG light chain, followed by a secondary anti-mouse IgG antibody (all from Cell Signalling) and ECL Prime Western detection kit (GE Healthcare).
5
Co-Immunoprecipitation of Signaling Proteins
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For co-immunoprecipitation analysis, cell-lysates were prepared using ice-cold HNGT buffer (50 mM HEPES, pH 7.5, 150 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Lysates from 20 × 106 purified primary human CD4+ CD27+ CD28+ T cells were incubated with monoclonal antibody to TAB1 or p38 (Cell Signalling) at 4 °C on a rotary shaker overnight. For the minor CD4+ CD28− CD27− subset, cells from two separate individuals were pooled to obtain sufficient cells for analysis. Extracts were then incubated with protein A–G conjugated agarose beads (Santa Cruz Biotechnology) at 4 °C for 3 h. Samples were washed and analyzed by western blotting as indicated. Co-immunoprecipitated proteins were detected using Mouse Anti-rabbit IgG Conformation Specific (L27A9; Cell Signalling) or Mouse Anti-rabbit IgG light chain, followed by a secondary anti-mouse IgG antibody (all from Cell Signalling) and ECL Prime Western detection kit (GE Healthcare).
6
Western Blot Analysis of Focal Adhesion Kinase
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OS cells were collected, and western blotting was performed as previously described [13 (link)]. Blots were blocked in TBS with 0.05% Tween 20 (Sigma-Aldrich) buffer (TBS-T) containing 1% Bovine Serum Albumin (BSA) (013-27054, Fujifilm), followed by incubation in TBS-T with 1% BSA and primary antibodies. Primary antibodies used were anti-FAK antibody (#13009, 1:1000, Cell Signaling Technology, MA, USA), anti-phospho-FAK antibody (44-624G, 1:1000, Invitrogen, MA, USA), anti-CXCR1 antibody (BS60349, 1:500, Bioworld, Bloomington, MN, USA), anti-CXCR2 antibody (20634-1 AP, 1:1000, Proteintech, IL, USA), and anti-β-actin antibody (A2228, 1:1000, Sigma-Aldrich). After washing thrice with TBS-T, membranes were incubated with secondary antibodies. Secondary antibodies used were anti-rabbit IgG antibody (#7074, 1:2000, Cell Signaling Technology) and anti-mouse IgG antibody (#7076, 1: 2000, Cell Signaling Technology). Images were captured using a ChemiDoc Touch and quantified using Image J software.
7
Protein Expression Analysis of CPE-Treated Cells
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The whole cell lysate from cells treated with CPE or vehicle control was isolated using MPER reagent along with inhibitors (1% phosphatase inhibitor, 1% protease inhibitor and 1% PMSF). BCA Assay was performed for measurement of protein concentration in the cell lysates. Equal amount of protein sample from vehicle control and CPE‐treated cells was separated using 12% or 15% SDS‐PAGE and then electrotransferred on to a PVDF membrane. The membranes were incubated (at 4°C) overnight, with primary antibody specific for NF‐κB, oxidative stress markers, LC3B, Beclin‐1, p62, BAD, Bcl‐2 (Abcam), BAX, p27 (Cell Signaling Technology) or GAPDH (Santa Cruz Biotechnology), followed by corresponding HRP conjugated anti‐rabbit IgG antibody (Cell Signaling Technology) or anti‐mouse IgG antibody (Cell Signaling Technology) incubation for 2 h. After that, the membranes were thoroughly washed in TBST (1×). Finally, the PVDF membrane was probed with ECL solution (Thermo Fisher) and observed in ChemiDoc Imaging System (Azure Biosystems) to visualize the protein bands.
8
Co-Immunoprecipitation of Signaling Proteins
9
Etomidate-Induced Oxidative Stress Regulation
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The following drugs and reagents were procured for the experiments: Etomidate (Jiangsu Enhua Pharmaceutical Co.); 0.9% sodium chloride injection (Zhejiang DuBang Pharmaceutical Co.); TRIzol (Takara Bio Inc.); complete protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd.); anti-Nrf2 protein antibody (Santa Cruz, USA); anti-HO-1 protein antibody (Santa Cruz, USA); anti-β-actin antibody (Santa Cruz Biotechnology, Inc.); PRIME-SCRIPT RT-PCR kit (Takara Bio Inc.); SYBR PreMix Ex kits (Takara Bio Inc.); tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 ELISA kits (Shanghai Westang Biotechnology Co., Ltd.); myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) kits (Nanjing Jiancheng Bioengineering Institute); and anti-mouse IgG antibody (Cell Signaling Technology, Inc.).
10
Protein Expression Analysis Protocol
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Protein was extracted using cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Protein content in the lysate was determined using the BCA Protein Assay (Pierce, Rockford, IL, USA). Equal amounts of protein were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to a polyvinylidene difluoride membrane. The following antibodies were used: matrix metalloproteinase (MMP)-1, SREBP-1, and Keratin 16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), nuclear factor kappa B (NF-kB) p65, interleukin (IL)-1α, IL-8, IL-6, collagen1, adiponectin, and phosphorylated insulin- like growth factor 1 receptor (Abcam, Cambridge, MA, USA), phospho ERK, Akt, and PI3K (Cell Signaling Technology), MMP-7, and MMP-12 (Thermo, Pittsburgh, PA, USA). Secondary anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG antibody (Cell Signaling Technology) were used to detect primary antibodies. Films of blots were analyzed and quantified using a densitometric program (TINA, Raytest Isotopenmebgerate, Straubenhardt, Germany). All experiments were repeated a minimum of four times.
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