Vinculin clone hvin 1
Manufactured by Merck Group
Sourced in United States
Vinculin (clone hVIN-1) is a laboratory reagent used in research applications. It is a monoclonal antibody that specifically recognizes the vinculin protein, which is involved in the anchoring of actin filaments to the cell membrane. The core function of this product is to enable the detection and analysis of vinculin in various experimental settings.
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6 protocols using vinculin clone hvin 1
1
Probing Actin Cytoskeleton Dynamics
2
Western Blot Analysis of Apoptosis Markers
3
Whole-Cell Protein Extraction for SDS-PAGE and Western Blot
4
Western Blot Analysis of Cell Signaling
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Cells were washed with phosphate-buffered saline (PBS) and lysed using RIPA buffer supplemented with protease and phosphatase inhibitors. Standard SDS–PAGE and Western blotting techniques were performed using PDVF membranes. Resulting membranes were then incubated in 5% bovine serum albumin (BSA) PBS for 1 h at room temperature before being incubated overnight in primary antibody solution in 5% BSA PBS. The following day, samples were rinsed in 0.2% Tween PBS and incubated for 45 min with HRP secondary antibody in 0.2% Tween-100× PBS. Samples were rinsed with PBS and imaged using chemiluminescence using a Bio-Rad ChemiDoc system. Primary antibodies used for Western blotting were anti-rabbit α-catenin (Sigma, catalogue# C2081), vinculin (clone hVIN-1, Sigma), and anti-mouse tubulin (clone DM1A, Cell Signaling).
5
Western Blot Antibody Sourcing
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Rabbit polyclonal antibodies against c-myc (A-14) and GFP (FL) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against GAPDH were purchased from Proteintech. Mouse monoclonal antibodies against vinculin, clone hVIN-1, were purchased from Sigma Aldrich (St. Louis, MO, USA). Mouse monoclonal antibodies against enterovirus VP1 (NCL-entero) were purchased from Novocastra (Buffalo Grove, IL, USA). XtremeGene HP plasmid transfection reagent was purchased from Sigma Aldrich. Brefeldin A (Invitrogen, Carlsbad, CA, USA) was dissolved in DMSO and used at a final concentration of 5 µg/mL. Also, 2-aminoethoxydiphenylborane (2APB, Sigma Aldrich) was diluted in DMSO and used at a final concentration of 100 µM.
6
Immunodetection Techniques for Cellular Analysis
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For immunoblotting, cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Roche). Proteins were subjected to SDS-PAGE. Immunoblotting was performed with antibodies against vinculin (clone hVIN-1, Sigma-Aldrich), p16INK4a (clone G175-405, BD Pharmingen).
For immunocytochemistry (ICC) and immunofluorescence (IF) analyses, cells were seeded on coverslips and fixed with ice cold methanol at −20°C for 15 minutes. Cells were then incubated with anti-p16INK4a antibody overnight at 4°C for ICC and 1 hour at room temperature for IF. Secondary antibodies with HRP or FITC conjugated were incubated and cells were analyzed using light microscope (Olympus) or with a bright-field immunofluorescence microscope (Zeiss). RAD51 (clone 14B4, Novus Biologicals) foci formation was stained and analyzed by in cell analyzer (BD Biosciences) as previously described [16 , 25 (link)].
For immunocytochemistry (ICC) and immunofluorescence (IF) analyses, cells were seeded on coverslips and fixed with ice cold methanol at −20°C for 15 minutes. Cells were then incubated with anti-p16INK4a antibody overnight at 4°C for ICC and 1 hour at room temperature for IF. Secondary antibodies with HRP or FITC conjugated were incubated and cells were analyzed using light microscope (Olympus) or with a bright-field immunofluorescence microscope (Zeiss). RAD51 (clone 14B4, Novus Biologicals) foci formation was stained and analyzed by in cell analyzer (BD Biosciences) as previously described [16 , 25 (link)].
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