Analysis of protein expression by immunoblotting was performed as previously described (10 (link)). Proteins of cell lysates (20–40 μg) were dissolved in SDS sample buffer, separated by 10% or 12.5% SDS-PAGE, and transferred onto a polyvinylidene fluoride membrane (Carl Roth GmbH, Karlsruhe, Germany). The membrane was blocked with 10% nonfat dry milk and incubated with the following primary antibodies: anti-ATGL, anti-HSL, anti-GAPDH, and anti-calnexin from Cell Signaling Technology (Danvers, MA; 2138S/ATGL, 4107S/HSL, 2118S/GAPDH, 2679S/calnexin), anti-6x HIS tag, and anti-NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) from Abcam (Cambridge, England; ab18184/6x HIS-tag®, ab157221/NDUFS1), anti-α-SMA from Thermo Fisher Scientific (Waltham, MA; PA5-22251/α-SMA), and anti-KIAA1363 from Invitrogen GmbH (PA5-50285/NCEH1 = KIAA1363), respectively. For detection, membranes were incubated with horseradish peroxidase-labeled secondary antibodies specific for respective primary antibody. Bands were visualized using the ECL plus Western blotting Detection Reagent (Thermo Fisher Scientific) and ChemiDoc Touch Imaging System (Bio-Rad).
Polyvinylidene fluoride membrane
Manufactured by Carl Roth
Sourced in Germany
Polyvinylidene fluoride (PVDF) membranes are a type of laboratory equipment used for various filtration and separation applications. They are made from a thermoplastic polymer material that exhibits high chemical resistance, thermal stability, and mechanical strength. PVDF membranes are commonly used for applications such as protein and nucleic acid separation, Western blotting, and sample preparation in analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc touch imaging system, by Bio-Rad (3 mentions)
Ecl plus prime, by GE Healthcare (2 mentions)
Rabbit anti phospho hsl s660, by Cell Signaling Technology (2 mentions)
Rabbit anti cgi 58, by Abnova (2 mentions)
Hrp linked sheep anti mouse antibody, by GE Healthcare (2 mentions)
Hrp linked rat anti rabbit antibody, by Agilent Technologies (2 mentions)
Ecl2 western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti hsl, by Cell Signaling Technology (2 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Anti calnexin, by Cell Signaling Technology (1 mentions)
Anti α sma, by Thermo Fisher Scientific (1 mentions)
Anti atgl, by Cell Signaling Technology (1 mentions)
Anti hsl, by Cell Signaling Technology (1 mentions)
Ecl plus western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Mouse anti his antibody, by GE Healthcare (1 mentions)
Anti lamp1, by Cell Signaling Technology (1 mentions)
Anti rab7, by Cell Signaling Technology (1 mentions)
Anti protein disulfide isomerase, by Cell Signaling Technology (1 mentions)
11 protocols using polyvinylidene fluoride membrane
1
Immunoblotting Analysis of Protein Expression
2
Western Blot Analysis of Protein Expression
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Proteins of tissue homogenates and cell extracts were separated by 10% SDS-PAGE according to standard protocols and blotted onto a polyvinylidene fluoride membrane (Carl Roth GmbH). Endogenous ABHD6 expression was detected using a rabbit polyclonal antibody raised against murine ABHD6 (14 (link)). His-tagged proteins were detected using mouse anti-His antibody (GE Healthcare/Amersham Biosciences). Cytochrome oxidase subunit 4 (CoxIV), protein disulfide isomerase, lysosomal-associated membrane protein 1 (LAMP-1), and ras-associated protein 7 (RAB7A) were detected using rabbit anti-COXIV, anti-protein disulfide isomerase, anti-LAMP-1, and anti-rab7 antibodies (all from Cell Signaling Technology). After binding of the secondary anti-mouse or anti-rabbit HRP-linked antibodies, signals were visualized by enhanced chemiluminescence detection (ECL Plus/Prime, GE Healthcare).
3
Western Blot Immunodetection of Proteins
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Proteins were separated by 10% SDS-PAGE according to standard protocols and blotted onto polyvinylidene fluoride membrane (Carl Roth GmbH). Membranes were blocked with 10% blotting grade milk powder (Roth) in TST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.4) at room temperature for 1 h or at 4°C overnight. Primary antibodies were directed against GST (GE Healthcare Cat# 27-4577-01, RRID:AB_771432), murine AOC3 (Abcam Cat# ab42885, RRID:AB_946102) and ZAG (Santa Cruz Biotechnology catalogue no. sc-11245, RRID:AB_2290216). Signals were visualized by enhanced chemiluminescence detection (Clarity Western ECL Substrate, Bio-Rad) and the ChemiDoc Touch Imaging System (Bio-Rad).
4
Western Blot Protein Expression Analysis
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Total protein extraction was performed using cell lysis buffer (Cell Signaling Technology, Frankfurt, Germany) according to the manufacturer's instructions. The protein concentration in the supernatant was determined with a Bradford assay. Western blotting was performed using 15 μg protein per sample, which were separated by SDS-polyacrylamide gel electrophoresis and transferred on a polyvinylidene fluoride membrane (Carl Roth, Karlsruhe, Germany) by electroblotting. Antibodies were incubated in 5% non-fat milk in TBST (0.1% Tween-20, 20 mM Tris, 140 mM NaCl, pH 7.6) overnight at 4°C. Proteins were detected with antibodies against p53 (DO-7, Dako Deutschland GmbH, Hamburg, Germany), p21 Waf1/Cip1 (12D1), Puma (D30C10), Gadd45a (D17E8), Bax (D2E11), CDK1 (8G10) (Cell Signaling Technology, Danvers MA, United States) and Mdm2 (SMP14, Santa Cruz, Heidelberg, Germany) using the biotechnology SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher, Rockford IL, USA).
5
SDS-PAGE and Western Blot Protocol
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Proteins were separated in 4%-12% SDS-PAGE gels (NuPAGE Bis-Tris, Invitrogen) and visualized using the Colloidal Blue Staining Kit (Invitrogen).
When used for western blots, proteins were transferred from the gel on a polyvinylidene fluoride membrane (Carl Roth GmbH) in CAPS transfer buffer (10 mM CAPS, 10% methanol, pH 11) . All antibodies were diluted as indicated in Supplemental Table S4 in 1× TST buffer (50 mM Tris, 0.1% Tween20, 0.15% NaCl, pH 7.4) with 1% milk powder. Detection of the chemiluminescence signal was performed with the Western blot Detection Reagent (Clarity Bio-Rad) on a ChemiDoc Touch Imaging System (Bio-Rad).
When used for western blots, proteins were transferred from the gel on a polyvinylidene fluoride membrane (Carl Roth GmbH) in CAPS transfer buffer (10 mM CAPS, 10% methanol, pH 11) . All antibodies were diluted as indicated in Supplemental Table S4 in 1× TST buffer (50 mM Tris, 0.1% Tween20, 0.15% NaCl, pH 7.4) with 1% milk powder. Detection of the chemiluminescence signal was performed with the Western blot Detection Reagent (Clarity Bio-Rad) on a ChemiDoc Touch Imaging System (Bio-Rad).
6
Western Blot Analysis of BLyS and BCMA
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40 µg of protein per sample were added to 4× loading buffer (bromophenol blue and Laemmli buffer), heated to 95°C for 5 min, and separated on a 12% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (Carl Roth GmbH, Karlsruhe, Germany). The following antibodies were used according to the instructions of the manufacturer: BLyS (ab16081; Abcam, USA), BCMA (ab5972, Abcam, USA). GAPDH (#2118, Cell Signaling, USA) and all secondary antibodies except anti-rat (ab102265, Abcam, USA) were purchased from Cell Signaling Technology (Frankfurt, Germany).
7
Western Blot Analysis of Protein Samples
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Proteins of cell extracts, tissue homogenates, and murine plasma (1/40 dilution) were separated by 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Carl Roth GmbH, Karlsruhe, Germany). The following primary antibodies were used: N-terminal His6 tag (27-4710-01, Amersham Biosciences, Buckinghamshire, UK), C-terminal His6 tag (102-PA80, Reliatech, Wolfenbüttel, Germany), and RBP4 (sc-25850, Santa Cruz Biotechnology Inc., Dallas, TX) as well as protein disulfide isomerase, LAMP1, and RAB7 from Cell Signaling Technology (35019, C54H11, and 2094S, respectively, Cambridge, UK). After binding of the secondary anti-mouse or anti-rabbit horseradish peroxidase-labeled antibody, signals were visualized by ECL detection (ECL Plus/Prime, GE Healthcare). For densitometric analysis, intensities of RBP4 bands were normalized to the corresponding intensities of the protein stain on the membrane using ImageJ software (freely available at http://imagej.nih.gov/ij/ ).
8
Western Blot Analysis of Lipid Metabolism Proteins
9
Western Blot Analysis of Lipid Metabolism Proteins
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Proteins of cell lysates or tissue homogenates were separated by SDS–PAGE according to their molecular weight using Tris/glycine as electrophoresis buffer and were transferred onto polyvinylidene fluoride membranes (Carl Roth GmbH, Karlsruhe, Germany) using CAPS buffer. After blocking, membranes were hybridized with respective primary antibodies. Membranes were washed, incubated with respective secondary horseradish-peroxidase (HRP)-conjugated antibody and detected using ECL2 Western blotting substrate (Thermo Scientific, Waltham, MA). Antibodies used were rabbit anti-HSL, rabbit anti-phospho-HSL (S660), and rabbit anti-GAPDH (all from Cell Signaling, Danvers, MA, USA), rabbit anti-CGI-58 (Abnova, Heidelberg), mouse anti-β-Actin (Santa Cruz, Santa Cruz, CA, USA), rabbit anti-alpha smooth muscle actin (α-SMA, Pierce, Thermo Scientific), HRP-linked sheep-anti mouse antibody (GE Healthcare Amersham, Buckinghamshire, UK), and HRP-linked rat-anti rabbit antibody (Dako, Glostrup, Denmark).
10
Western Blot Analysis of sFRP5
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A Bradford assay was conducted and the protein assay dye reagent (BIO-RAD; Ca, USA) was used. Protein (30 µg per sample) was added to 4 × loading buffer (bromophenol blue and Laemmli buffer), heated to 95 °C for 5 min, and separated on a 12.5% SDS-PAGE gel. Afterwards, proteins were transferred to polyvinylidene fluoride membranes (Carl Roth, Karlsruhe, Germany). The following antibodies were used according to the instructions of the manufacturer: sFRP5 (Santa Cruz Biotechnology; Dallas, Texas; USA) and secondary antibody anti-rabbit (Cell signalling; Danvers, Massachusetts, USA). Membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific; Waltham, Massachusetts, USA) for 5 min at room temperature and the signals were detected with the Molecular Imager ChemiDoc XRS+ (Bio Rad; Hercules, California, USA).
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