LPS samples were diluted in PBS and applied to a PVDF membrane. Unspecific binding sites were blocked with TBS/0.05% Tween-20/5 mM CaCl2/3% BSA for 1 h at RT. All washing and incubation steps were performed with TBS/0.05% Tween-20/5 mM CaCl2. To detect ANXA5 binding, membranes were incubated with 2 μg/ml ANXA5 (Sigma, A9460) at 4°C overnight. After washing three times, the primary antibody (S3 Table ) was incubated for 4 h at RT to detect ANXA5 binding. For detection of Lewis Y or lipid A (S3 Table ), membranes were incubated with the respective primary antibodies at 4°C overnight. Blots were incubated with HRP-coupled secondary antibodies for 1 h at RT and detection was performed using Immobilon Chemiluminescent HRP Substrate (Millipore).
Anxa5
Manufactured by Merck Group
Sourced in United States
ANXA5 is a protein that can be used in laboratory settings. It functions as a calcium-binding protein and is involved in various biological processes. The core function of ANXA5 is to bind to phospholipids in a calcium-dependent manner. This property can be utilized in various laboratory applications, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared fluorescent system, by LI COR (2 mentions)
Plastic tissue culture flasks, by Corning (2 mentions)
Gel image analysis system, by Bio-Rad (2 mentions)
Digital image analysis system, by Nikon (2 mentions)
Ultraviolet analyzer, by Beckman Coulter (2 mentions)
E cadherin, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Proliferating cell nuclear antigen (pcna), by Merck Group (2 mentions)
P erk1 2, by Merck Group (2 mentions)
Mmp 9, by Merck Group (2 mentions)
Pmek1 2, by Merck Group (2 mentions)
Anxa5 protein, by Novoprotein (2 mentions)
Sirna targeted to anxa5, by Thermo Fisher Scientific (2 mentions)
Mek1 2, by Merck Group (2 mentions)
Erk1 2, by Merck Group (2 mentions)
Bcl 2, by Merck Group (2 mentions)
Bcl2 associated x (bax), by Merck Group (2 mentions)
Gapdh, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
5 protocols using anxa5
1
Annexin A5 Binding Assay
2
Annexin A5 Binding to Trophoblasts
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Plasma membrane protein fractions from cytotrophoblasts or syncytiotrophoblasts were resolved by SDS-PAGE and blots were incubated for 16 h at 4 °C with 0.5 μM of AnxA5-CF770 (Biotium, France) alone or in presence of an excess of unlabeled recombinant AnxA5 (1 μM; Sigma-Aldrich, France). Bound AnxA5-CF770 was visualized using Odyssey infrared fluorescent system (Li-Cor, France).
3
Plasma Membrane Protein Extraction and Analysis
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Total cell extracts were prepared as previously described by41 (link). Plasma membrane protein fractions were obtained using the ProteoExtract Native Membrane protein Extraction Kit (Merck Millipore, France) accordingly do the manufacturer’s protocol. Protein samples were resolved by SDS-PAGE and immunoblotted with antibodies to AnxA1, AnxA2, AnxA6 (0.2 μg/ml each, Santa-Cruz Biotechnology, Germany), AnxA5 (2 μg/ml, Sigma-Aldrich, France), α-Catenin (0.5 μg/ml, Life Technologies, France), β-Catenin (0.25 μg/ml, Life Technologies, France), E-Cadherin (0.25 μg/ml, Life Technologies, France), ezrin (0.5 μg/ml, Life Technologies, France), actin (0.8 μg/ml, Sigma-Aldrich, France), or GFP (1 μg/ml, Clontech, France). After incubation with appropriate DyLight Fluor-conjugated secondary antibody (680 or 800 conjugate, Life Technologies, France) blots were revealed by using Odyssey infrared fluorescent system (Li-Cor, France). Conversely, blot with cell membrane factions were incubated with reversible protein stain (Thermo scientific, France).
4
Gastric Cancer Cell Lines Cultivation
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Human gastric cancer cell lines MGC-803 and MKN-45 were obtained from Shanghai Academy of Medical Sciences (Shanghai, China). The cells were incubated at 37°C in 5% CO2 in plastic tissue culture flasks (Corning Inc., Acton, MA, USA) with complete Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL). ANXA5, PCNA, Bcl-2, Bax, MMP-9, E-cadherin, β-actin, GAPDH, MEK1/2, pMEK1/2, ERK1/2, and pERK1/2 monoclonal antibody were purchased from Sigma (St. Louis, MO, USA) and the ANXA5 protein was bought from novoprotein (Shanghai, China); the Trizol reagent, protein assay kit, the siRNA targeted to ANXA5 and the negative control siRNA were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA); the gel image analysis system and electrophoresis apparatus were purchased from Bio-Rad (Hercules, CA, USA). The digital image analysis system and the fluorescence microscope were from Nikon (Tokyo, Japan), and the ultraviolet analyzer was from Beckman Coulter (Miami, FL, USA).
5
Gastric Cancer Cell Lines Cultivation
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Human gastric cancer cell lines MGC-803 and MKN-45 were obtained from Shanghai Academy of Medical Sciences (Shanghai, China). The cells were incubated at 37°C in 5% CO2 in plastic tissue culture flasks (Corning Inc., Acton, MA, USA) with complete Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL). ANXA5, PCNA, Bcl-2, Bax, MMP-9, E-cadherin, β-actin, GAPDH, MEK1/2, pMEK1/2, ERK1/2, and pERK1/2 monoclonal antibody were purchased from Sigma (St. Louis, MO, USA) and the ANXA5 protein was bought from novoprotein (Shanghai, China); the Trizol reagent, protein assay kit, the siRNA targeted to ANXA5 and the negative control siRNA were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA); the gel image analysis system and electrophoresis apparatus were purchased from Bio-Rad (Hercules, CA, USA). The digital image analysis system and the fluorescence microscope were from Nikon (Tokyo, Japan), and the ultraviolet analyzer was from Beckman Coulter (Miami, FL, USA).
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