Cell lysis and immunoblotting were essentially performed as described previously [8 (link),9 (link),10 (link)] with minor modifications. Proteins were fractionated by polyacrylamide gel electrophoresis on TGX Stain-Free FastCast gels (BioRad, Munich, Germany). Following blotting and antibody treatment, chemoluminescent detection of proteins was carried with a ChemiDoc XRS+ System with Image Lab Software (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to either the total amount of protein in the same lane (when using the TGX Stain-Free FastCast gels), or to bands for the housekeeping gene GAPDH. Significant differences (p < 0.05)-calculated with the unpaired two-tailed Student’s t test–denoted by bars above the respective cell lines. The antibodies used were Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), E-cadherin (#610181) and Rac1 (#610650) (BD Transduction Laboratories, Heidelberg, Germany), GAPDH (14C10, #2118, Cell Signaling Technology, Frankfurt am Main, Germany), Vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), and HA (clone 12CA5, Roche Diagnostics, Mannheim, Germany).
Vimentin clone v9
Manufactured by Merck Group
Sourced in Germany
Vimentin (clone V9) is a laboratory reagent used for the detection and analysis of the intermediate filament protein vimentin. Vimentin is a structural protein found in various cell types and is commonly used as a marker for certain cell lineages and disease states. The V9 clone is a specific monoclonal antibody that binds to vimentin and is often used in immunohistochemical and immunocytochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Tgx stain free fastcast gels, by Bio-Rad (2 mentions)
E cadherin, by BD (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Chemidoc xrs system with image lab software, by Bio-Rad (1 mentions)
Rac1b, by Merck Group (1 mentions)
Ha clone 12ca5, by Roche (1 mentions)
Amersham ecl prime detection reagent, by GE Healthcare (1 mentions)
Phospho smad2 s465 s467, by Cell Signaling Technology (1 mentions)
Smad2, by Abcam (1 mentions)
Mini protean tgx any kd precast gels, by Bio-Rad (1 mentions)
Ferritin, by Merck Group (1 mentions)
Map2 clone hm 2, by Merck Group (1 mentions)
Nf clone nf 01, by Exbio (1 mentions)
Secondary fl uorescein and rhodamineconjugated antibodies, by Merck Group (1 mentions)
3 protocols using vimentin clone v9
1
Cell Lysis and Immunoblotting Protocol
2
Western Blot Procedure for Protein Analysis
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The Western blot procedure was performed as described in detail earlier [19 (link)]. Briefly, equal amounts of crude proteinaceous extracts from each cell line (three preparations harvested at different times during the continuous culture) were fractionated by SDS-PAGE on mini-PROTEAN TGX any-kD precast gels or TGX Stain-Free FastCast gels (BioRad, Munich, Germany) and blotted to PVDF membranes. The membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies to E-cadherin (#610181, BD Transduction Laboratories, Heidelberg, Germany) or vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), phospho-Smad2 (S465/S467) (#3101, Cell Signaling Technology, Frankfurt/Main, Germany), Smad2 (#1736-1, Epitomics, Burlingame, CA, USA), and GAPDH (Cell Signaling Technology) as loading controls.
3
Identifying Brain Cell Types
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To identify astroglia cell types, we used antibodies against GFAP (clone GF-01, 1:100, Exbio, Prague), and polyclonal sera to GFAP (1:100, Dako). Oligodendroglia cells were detected by antibodies to GalC (clone mGalC, 1:10, Boehringer-Mannheim, Vienna) and to O4 antigen (clone 81, 1:10, Boehringer-Mannheim, Vienna). Microglial cells were defi ned as immunoreactive with antibodies to CD11c (clone BU15, 1:50, Immunotech, France), and polyclonal sera to ferritin (1:50, Sigma). Neuronal cells were identifi ed with antibodies to MAP2 (clone HM-2, 1:50, Sigma), and NF (clone NF-01, 1:100, Exbio, Prague). The endothelial cells were detected by polyclonal sera against Von Willebrand Factor-VIII-related antigen (1:100, Dako). The antibodies to vimentin (clone V9, 1:100, Sigma), to cytokeratins monoclonal anti-pan CK (types: 1,4,5,6,8,10,13,18,19, 1:100, Sigma), and polyclonal sera against fi bronectin (1:100, Sigma) were used for further characterization of human brain cells. The secondary fl uorescein-and rhodamineconjugated antibodies were purchased from Sigma and Sevapharma (Prague).
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