Cellular protein samples were isolated with a Protein Extraction Kit (Qiagen, GmbH, Hilden, Germany) according to the kit's manual and supplemented with a protease inhibitor cocktail (Abcam, Cambridge, UK). Protein samples were separated with 10% SDS-PAGE gel and were transferred to a polyvinylidene fluoride hydrophobic membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skimmed milk (Solarbio, Beijing, China) overnight at 4°C to cover the nonspecific binding sites. Then the membrane was inoculated with the rabbit anti-mouse KLF-4 (BM0485, Abzoom Biolabs, Dallas, TX, USA; 1 : 300), PAI-1 (sc-8979, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), E-cadherin (sc-7870, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 500), Collagen I (ab21286, Abcam, Cambridge, UK; 1 : 200), mouse anti-mouse α-Smooth Muscle Actin (α-SMA) (sc-53142, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), or rabbit anti-mouse β-actin (sc-7210, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 800) at room temperature for 2 hours. The specific binding to each protein marker was presented with the incubation with the peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA) and the electrochemiluminescence (ECL) detection system (Amersham, Uppsala, Sweden). The protein level was presented as a ratio to β-actin.
Polyvinylidene fluoridehydrophobic membrane
Manufactured by Merck Group
Sourced in United States
Polyvinylidene fluoride (PVDF) is a hydrophobic membrane material. It is a synthetic fluoropolymer with a high chemical and thermal resistance. The PVDF membrane exhibits a hydrophobic surface that repels water and aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Abcam (3 mentions)
Protein extraction kit, by Qiagen (1 mentions)
Skimmed milk, by Solarbio (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Sc 7210, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated secondary antibody, by Promega (1 mentions)
Sc 8979, by Santa Cruz Biotechnology (1 mentions)
Sc 53142, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Nuclear cytosol fractionation kit, by Abcam (1 mentions)
Enhanced chemiluminescence detection, by GE Healthcare (1 mentions)
Gapdh, by Sino Biological (1 mentions)
Horseradish peroxidase linked secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mitochondria cytosol fractionation kit, by Abcam (1 mentions)
Antibody against gapdh, by Abcam (1 mentions)
5 protocols using polyvinylidene fluoridehydrophobic membrane
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of HO-1 and STAT3
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Protein samples were extracted from placental tissues or JEG-3 cells with NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce, Rockford, IL, USA). These protein samples were separated by electrophoresis using a 12% SDS-PAGE gel and then were transferred to a polyvinylidene fluoride hydrophobic membrane (Millipore, Bedford, MA, USA). Then the membrane was blocked with 2% BSA (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C and was incubated with rabbit polyclonal antibody against human HO-1 and STAT3 with or without phosphorylation (Tyr 705) or β-actin respectively. Finally, HRP-conjugated secondary antibody against rabbit IgG and electrochemiluminescence (ECL) (Amersham, Uppsala, Sweden) were used to visualize the protein bands. The levels of HO-1 and STAT3 with or without phosphorylation (Tyr 705) were presented as a percent gray value normalized to β-actin.
3
Quantifying Sam68 levels in cancer
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Cellular protein samples were extracted from cancer tissues or OVCAR-3 cells with Nuclear/Cytosol Fractionation Kit (BioVision, San Diego, CA, USA), separated via 10% SDS-PAGE electrophoresis, and then transferred onto polyvinylidene fluoridehydrophobic membrane (Millipore, Bedford, MA, USA). Western blotting was performed using rabbit polyclone antibody against human Sam68 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit polyclone antibody against β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Horseradish peroxidase-linked goat anti-rabbit IgG (Pierce, Rockford, IL, USA) and the enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA) were utilized to analyze Sam68 level in cancer tissues or OVCAR-3 cells.
4
Quantification of Smad 3 in MC3T3-E1 Cells
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The cytoplasmic protein sample from the treated MC3T3-E1 cells was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce, Rockford, IL, USA) according to the product's manual, and was supplemented with Protease Inhibitor Cocktail (Abcam, Cambridge, UK). Protein samples were separated with 12% SDS-PAGE, and were transferred to a polyvinylidene fluoridehydrophobic membrane (Millipore, Bedford, MA, USA). The membrane was incubated with the primary antibodies against Smad 3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or against GAPDH (Sinobiological, Beijing, China), and then was incubated with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) successively. Four-washings with 1x Phosphate Buffered Saline Tween-20 (PBST) were performed before each incubation. The specific quantification of Smad 3 in the membrane was colorized with enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Uppsala, Sweden).
5
Mitochondrial Fractionation and Protein Analysis
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Mitochondria/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA) was utilized to isolate the mitochondrial and cytosolic protein sections in EOC OVCAR-3 cells under the guidance of the kit's manual. Protein samples were centrifuged with 12,000 x g for 15 min to remove cell debris, and then were supplemented with Protease Inhibitor Cocktail (Abcam, Cambridge, UK) (ab65621) and were stored at -80°C before use. To perform western blotting, samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred to a polyvinylidene fluoridehydrophobic membrane (Millipore, Bedford, MA, USA). Rabbit polyclone antihuman galectin-3, cytochrome c, caspase 3 and caspase 9 antibodies (1:1000 respectively, Abcam) were utilized to incubate the membrane at 4°C overnight, with the antibody against GAPDH (1:1000 respectively, Abcam) as internal control. Goat anti-rabbit IgG conjugated horseradish peroxidase (1:2000, Bio-Rad Laboratories, Hercules, CA, USA) and the enhanced chemiluminescence (ECL) (Thermo Scientific, Rockford, IL, USA) were utilized to visualize the specific antigen-antibody binding.
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