Proteins extraction from cells and tissues was performed using RIPA buffer, and then the extracted proteins separated by gel electrophoresis with a 10% SDS‐PAGE gel. Separated proteins were then transferred onto polyvinylidene difluoride membranes. The membranes were then incubated with 5% skimmed milk for a duration of 2 hours at room temperature with agitation, after that, the membranes were incubated with ANXA2 primary antibody (1:1000; ab41803; Abcam) or β‐actin primary antibody (1:2000; ab8227, Abcam) at 4°C overnight, followed by incubating with relevant secondary antibodies with horse‐radish peroxidase conjugation (1:2000; Santa Cruz Biotechnology, Dallas) for 2 hours at room temperature. Western blot bands were detected using an enhanced chemiluminescence kit (Pierce, Rockford, USA).
Ab41803
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab41803 is an antibody raised against a specific target. It is suitable for use in various laboratory applications. The antibody's core function is to bind to and detect the target antigen. No further details about the intended use or performance of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab8227, by Abcam (3 mentions)
Criterion tgx precast gel, by Bio-Rad (2 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (2 mentions)
Ab9484, by Abcam (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab137321, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Difco skim milk, by BD (1 mentions)
X ray film, by Thermo Fisher Scientific (1 mentions)
Ecl method, by Thermo Fisher Scientific (1 mentions)
Protein g agarose beads, by Merck Group (1 mentions)
Ab6721, by Abcam (1 mentions)
Hpa006881, by Merck Group (1 mentions)
Hpa000931, by Merck Group (1 mentions)
Clone dm1a, by Merck Group (1 mentions)
Anti mouse alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
4 6 diamino 2 phenyl indole dapi, by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
27 protocols using ab41803
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Cellular Proteins
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Samples were separated by 8–16% SDS-PAGE and transferred to nitrocellulose membrane (0.45 μm). 5% Difco skim milk (BD Bioscience) in PBST (0.05% Tween 20) was incubated for blocking, and the membranes were applied with specific antibodies as described in the previous materials section. After incubation with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG (Amersham Bioscience), the antigen-antibody was detected in X-ray film (AGFA) by an ECL method (Thermo Scientific). Primary antibodies used include the following: rabbit anti-Vimentin (ab137321, Abcam), rabbit anti-GFAP (ab7260, Abcam), rabbit anti-FXR2 (#7098, Cell Signaling), rabbit anti-Annexin2 (ab41803, Abcam) and rabbit anti-beta-Actin HRP conjugate (13E5, Cell Signaling). The western blots were quantified using ImageJ and obtained by averaging the data from three independent experiments. Statistical significance was determined by unpaired student’s t-test using GraphPad prism software.
3
S-Nitrosylation of Annexin II in JEG-3 Cells
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JEG-3 cells (2.5×105) were treated with 10 nM ADM or 100 μM S-Nitrosoglutathione (GSNO), an endogenous nitrogen oxide, for 24 h. The concentrations of ADM and GSNO used were based on our previous studies indicating their biological effects in the in vitro study [7 (link)]. Cells treated with PBS were used as a control. S-nitrosylated proteins were purified and resolved by SDS-PAGE. S-nitrosylated annexin II (ANX II) was detected by western blot using a polyclonal anti-ANX II antibody (1:1000; ab41803; Abcam).
4
Immunoprecipitation of Annexin A2 and Fibronectin
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For Annexin A2 immunoprecipitation, cell lysates were pre-cleared with protein G agarose beads (Sigma Aldrich) for 1 hour with gentle rotation at 4°C. Cleared lysates were then transferred in a new tube for incubation with agarose beads and primary anti-Annexin A2 antibody (Rabbit, ab41803, Abcam) overnight at 4°C with gentle rotation. The beads were washed with lysis buffer and boiled with 2x SDS loading buffer at 100°C for 5 minutes before separation using SDS-PAGE.
For fibronectin immunoprecipitation, protein G agarose beads were incubated with anti-FN for 1 hour, rotating at 4°C. Beads were then spun down and added to purified fibronectin-PBS solution for 3 hours, rotating at 4°C. Beads were then washed with lysis buffer and added to MDA-MB-231 cell lysate, and incubated overnight, rotating at 4°C. The beads were washed with lysis buffer and boiled with 2x SDS loading buffer at 100°C for 5 minutes before separation using SDS-PAGE. For all experiments, a control/blank IP was prepared with all the reagents, but without the antibody, to account for non-specific binding.
For fibronectin immunoprecipitation, protein G agarose beads were incubated with anti-FN for 1 hour, rotating at 4°C. Beads were then spun down and added to purified fibronectin-PBS solution for 3 hours, rotating at 4°C. Beads were then washed with lysis buffer and added to MDA-MB-231 cell lysate, and incubated overnight, rotating at 4°C. The beads were washed with lysis buffer and boiled with 2x SDS loading buffer at 100°C for 5 minutes before separation using SDS-PAGE. For all experiments, a control/blank IP was prepared with all the reagents, but without the antibody, to account for non-specific binding.
5
Annexin A2 Extraction and Detection
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MPC5 cells were pre-treated with 20 μg/mL Annexin A2 antibody after 12 hours of synchronization. The cells were immediately equilibrated to 4 °C on ice for 5 minutes before being rinsed with ice-cold PBS and treated with 10 mM EGTA in PBS (600 μL/6 cm dish) for 30 minutes. The eluates were collected after centrifugation at 500 g for 10 minutes. Total protein extracts were quantified using a BCA protein assay kit (#23227, ThermoFisher) before being separated by 10% SDS-PAGE and then transferred to PVDF membranes (250 mA/2.5 h). After being blocked with 5% non-fat milk for 1 hour at room temperature, the membranes were incubated with primary Annexin A2 antibody (1 μg/mL; #ab41803, Abcam) and then with secondary HRP-conjugated goat anti-rabbit IgG antibody (1:10,000; #ab6721, Abcam). Signals were detected with an enhanced chemiluminescence (ECL) kit (#34095, ThermoFisher). Images were captured using the Bio-Rad Imaging System.
6
Immunofluorescence Staining of Cellular Proteins
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Cells were seeded in 6-well plates containing coverslip in complete growth medium and, after treatment, fixed in 4% paraformaldehyde for 20 min at 20 °C, and permeabilized in 0.5% v/v Triton X-100 in PBS for 10 min, prior to be blocked with 5% v/v goat serum and 0.1% v/v Triton X-100 in PBS for 1 h. For ANXA2, permeabilization and blocking were performed with 0.2% w/v saponin in presence of 5% v/v goat serum and 0.2% w/v BSA in PBS, for 1 h. Immunofluorescence staining was obtained using anti-annexin A2 (1:125, ab41803, Abcam, Cambridge, UK), anti-NDRG1 (1:50, HPA006881, Sigma-Aldrich), anti-PARP1 (1:800, 46D11, Cell Signaling Technology, Danvers, MA), anti-STAT1 (1:100, HPA000931, Sigma-Aldrich) and anti-α-tubulin (1:200; clone DM1A, Merck Millipore, MA, USA), following overnight incubation at 4 °C. After washing, cells were incubated with secondary antibody anti-mouse Alexa Fluor-488 conjugate or anti-rabbit Alexa Fluor-488 and Alexa Fluor-567 conjugate (1:200) (Thermo Fisher Scientific), in the dark for 30 min at 20 °C. Coverslip was mounted onto slides using an antifade mounting reagent containing DAPI. Slides were observed under a fluorescence microscope (Leica Biosystems, Newcastle, UK) using a 100X oil immersion objective.
7
Immunofluorescence Imaging of AnxA2 in PC12 Cells
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PC12 cells were grown on poly-L-Lys-coated glass coverslips and treated as indicated above. The cells were fixed, permeabilised and blocked against non-specific binding of antibodies as described previously (Grindheim et al., 2014 (link); Grindheim et al., 2016 (link)) prior to staining with primary polyclonal antibodies against AnxA2 (1:250; ab41803, Abcam, Cambridge, UK). The bound primary antibodies were detected using appropriate DyLight-488- or DyLight-594-conjugated Fab2 fragments (1:50; Jackson ImmunoResearch Laboratories, West Grove, United States). The coverslips were inverted and mounted on objective glasses on a small drop of Vectashield mounting medium containing 4′,6-diamino-2′-phenylindole (DAPI) (Vector Laboratories, Burlingame, United States). Confocal imaging was performed using a Leica SP5 AOBS confocal laser scanning microscope equipped with 405 diode and argon and helium neon lasers (Leica Microsystems, Wetzlar, Germany). Optical sections were obtained using the 63×/1.4 NA HCX Plan-Apochromat oil-immersion objective (Leica, Wetzlar, Germany), ∼1 Airy unit pinhole aperture and appropriate filter combinations. Confocal images were obtained in Leica Application Suite (LAS) AF. Figures were made in Microsoft Publisher for the images and GraphPad Prism for the graphs. Quantitation was done with ImageJ and transferred to GraphPad.
8
Extracellular Vesicle Characterization and HDAC Activity
9
Western Blot Analysis of Tumor Proteins
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Protein extracts from tumor tissue samples were prepared by suspending the cells in a cell lysis buffer (Sigma) and protease inhibitor cocktail (Roche). Each extract was prepared as above and an equivalent to 20 μg total protein was separated by SDS-PAGE.
Annexin II Antibody (ab41803, Abcam©, France); hENT1 (antibody LS-C178673, LSBio©, France); AKT (ab8932, Abcam©, France); ERK-p (9101S Cell signaling, France); Raf-1 (ab173539, Abcam©, France); mTOR (ab1093, Abcam©, France); Caspase-3 (ab47131, Abcam©, France). Horseradish peroxidase-conjugated anti-mouse (Promega W402B 28570702©, France) or anti-rabbit IgG (w401B 29303402© Promega, France) was used to detect specific proteins.
Detection of specific proteins was carried out using an enhanced chemiluminescence western blotting kit (Pierce).
We measured the intensity of each band using LAS 4000 software and calculated the relative protein levels normalized to that of the β-actin antibody (A5316-2ML SIGMA ©, France).
Annexin II Antibody (ab41803, Abcam©, France); hENT1 (antibody LS-C178673, LSBio©, France); AKT (ab8932, Abcam©, France); ERK-p (9101S Cell signaling, France); Raf-1 (ab173539, Abcam©, France); mTOR (ab1093, Abcam©, France); Caspase-3 (ab47131, Abcam©, France). Horseradish peroxidase-conjugated anti-mouse (Promega W402B 28570702©, France) or anti-rabbit IgG (w401B 29303402© Promega, France) was used to detect specific proteins.
Detection of specific proteins was carried out using an enhanced chemiluminescence western blotting kit (Pierce).
We measured the intensity of each band using LAS 4000 software and calculated the relative protein levels normalized to that of the β-actin antibody (A5316-2ML SIGMA ©, France).
10
Immunofluorescent Localization of AnxA2 and Phosphorylated Forms
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PC12 cells were fixed, permeabilised and blocked as described previously 16 , 61 , prior to staining with primary antibodies against AnxA2 (polyclonal; ab41803; Abcam, 1 : 250 dilution), pTyr23AnxA2 (monoclonal; sc‐135753; Santa Cruz Biotechnologies; 1 : 20 dilution), pSer25AnxA2 (polyclonal; OAAF00618; Aviva Systems Biology; 1 : 250 dilution) and GW182 (monoclonal; sc‐56314; Santa Cruz Biotechnologies; 1 : 10 dilution). The bound primary antibodies were detected using appropriate DyLight 488‐ or DyLight 594‐conjugated goat anti‐rabbit or ‐mouse Fab2 fragments (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1 : 50 dilution).
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