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6 protocols using tgx stain free fastcast gels

1

Cell Lysis and Immunoblotting Protocol

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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).
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2

Cell Lysis and Immunoblotting Protocol

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Cell lysis and immunoblotting was essentially performed as described previously [42 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with PhosphoSafe lysis buffer (Merck Millipore, Darmstadt, Germany). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by PAGE on mini-PROTEAN TGX any-kD precast gels, or TGX Stain-Free FastCast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies (CgA: Monoclonal Mouse Anti-Human Chromogranin A, clone DAK-A3, Dako, Glostrup, Denmark; SYP: Anti-Synaptophysin, Dako; SSTR2: Anti-Somatostatin Receptor 2 antibody [UMB1]-C-terminal #ab134152, Abcam, Cambridge, UK) overnight at 4 °C. After washing and incubation with horseradish peroxidase-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS imaging system (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to those for the housekeeping genes GAPDH or HSP90.
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3

Cell Lysis and Immunoblotting Protocol

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Cell lysis and immoblotting was essentially performed as described previously [22 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with 1× PhosphoSafe lysis buffer (Merck Millipore). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by polyacrylamide gel electrophoresis on mini-PROTEAN TGX any-kD precast gels, or TGX Stain-Free FastCast gels (BioRad) and blotted to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies either for 2 h at RT or overnight at 4 °C. After washing and incubation with HRP-linked secondary antibodies, chemoluminescent detection of proteins was done on 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.
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4

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.
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5

Cell Lysis and Immunoblotting Protocol

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Cell lysis and immunoblotting were essentially performed as described previously [13 (link),18 (link),19 (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 done on a ChemiDoc XRS imaging system (BioRad, Munich, Germany) with an Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The antibodies used were: ALK2: Biorbyt Ltd., Cambridge, UK; phospho-SMAD1/5 and SMAD1: Cell Signaling Technology (CST), Frankfurt/Main, Germany; RUNX3/AML2: D9K6L, CST; RAC1: #610650, BD Biosciences, Heidelberg, Germany; RAC1b: #09-271, Merck Millipore, Darmstadt, Germany; GAPDH: 14C10, CST; HSP90: #13119, Santa Cruz Biotechnology, Heidelberg, Germany. The signals for the proteins of interest were normalized to the total amount of protein in the same lane, and significant differences (p < 0.05) were calculated with the unpaired two-tailed Student’s t test.
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6

Protein Extraction and Western Blot Analysis

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Proteins were extracted from livers by homogenizing in modified radio-immunoprecipitation (RIPA) assay buffer (Thermo Fisher Scientific Inc.). Protein was loaded in equal amounts per lane and separated using TGX Stain-Free™ FastCast™ Gels (Bio-Rad) of appropriate gradients and transferred to a polyvinylidene fluoride (PVDF) membrane using Immobilon-FL Transfer Membranes (MilliporeSigma, Burlington, MA, USA). The PVDF membrane was blocked using Blocker™ FL Blocking Buffer (Thermo Fisher Scientific Inc.) for an hour followed by incubation with primary antibodies for fatty acid synthase (FASN) (Santa Cruz Biotechnology, CA, USA; dilution 1:1000) and phosphorylated (Thr172) [27 (link)] and total AMP-activated protein kinase (AMPK) (Cell Signaling Technologies, Danvers, MA, USA; dilution 1:500). Protein concentrations were normalized to TATA-Box binding protein (TBP; Cell Signaling Technologies; dilution 1:1000). Mouse polyclonal antibody was used as a secondary antibody for FASN (dilution 1:25,000), and rabbit polyclonal antibody was used as a secondary antibody for AMPK (dilution 1:25,000).
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