Total cell proteins were isolated with a total protein extraction kit (Keygen). Concentration of proteins was determined with the BCA protein assay kit (Cowin BioTech, Beijing, China). 30μg of protein was loaded and separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). The antibody against OLFM4 (Lifespan, rabbit anti-human polyclonal, Seattle, USA) was used to analyze protein expression. Proteins were visualized using horseradish peroxidase-conjugated secondary antibody. Signal was detected by enhanced chemoluminescence techniques (Millipore). Detection of GAPDH with specific antibody (Cell Signaling Technology) was used as the loading control.
Polyvinylidine difluoride membrane
Manufactured by Merck Group
Sourced in United States
Polyvinylidine difluoride membrane is a type of laboratory filtration membrane. It is a synthetic polymer material with a high chemical and thermal resistance. The membrane is designed to filter and separate various substances during analytical and preparative procedures in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
γ h2ax, by Abcam (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
α actinin, by Santa Cruz Biotechnology (3 mentions)
Twist, by Santa Cruz Biotechnology (3 mentions)
Enhanced chemoluminescence techniques, by Merck Group (2 mentions)
Bca protein assay kit, by CoWin Biotech (2 mentions)
Bradford method, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Opcml, by Abcam (1 mentions)
Phospho protein kinase b akt ser473, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Anti p16, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Donkey anti goat igg hrp, by Santa Cruz Biotechnology (1 mentions)
42 protocols using polyvinylidine difluoride membrane
1
OLFM4 Protein Expression Analysis
2
Protein Expression Analysis in Cells
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Total protein of cells was extracted by lysis buffer and concentration determination was conducted using the DC protein assay method of Bradford (Bio-Rad, Hercules, CA). Twenty mg of protein was loaded and separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). The antibodies used for the analysis are as follows, OPCML (Abcam, Cambridge, UK), cyclin-dependent kinase inhibitor 1B (p27), cleaved caspase 3, cleaved caspase 9, poly ADP-ribose polymerase (PARP), phospho-Protein Kinase B (AKT) (Ser473) and phosphor-glycogen synthesis kinase 3β (GSK3β) (Cell Signaling Technology, Inc., Danvers, MA), and GAPDH (Good Here Biotechnology, Hangzhou, P.R. China).
3
Immunoprecipitation and Detection of p53
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Cells were harvested in lysis buffer (50 mM Tris (pH 8.0), 5 mM NaCl, 0.5% NP-40 and 1x protease inhibitor) and freeze/thawed three times, and the protein was recovered. Protein concentrations were determined using the Bradford method (Bio-Rad, CA, USA). Cell extracts containing equivalent amounts of protein were immunoprecipitated in lysis buffer containing the indicated monoclonal antibody against p53 overnight (4°C). Protein A/G Sepharose beads were added to the immunoprecipitation mixture for 1 hr before three washes with SNNTE buffer (5% sucrose, 1% NP-40, 0.5 M NaCl, 50 mM Tris (pH 7.4) and 5 mM EDTA). The entire immunoprecipitate was then suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled, and loaded onto an SDS-polyacrylamide gel. The separated proteins were transferred onto polyvinylidine difluoride membranes (Millipore, USA) and detected using antibodies against HA (3 F10, Hoffmann-La Roche, Switzerland), p53 conformation (Pab246, Calbiochem, USA) and p53N, p53C, p21 and actin (Santa Cruz Biotechnology, USA).
4
Protein Extraction and Western Blot Analysis
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The cells were lysed in lysis buffer (50 mM Tris–HCl, 120 mM NaCl, 0.5% NP-40, 100 mM NaF, 200 µM Na3VO4 and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland)) for 30 min at 4°C. The lysates were centrifuged at 13,000× g for 10 min at 4°C. After determining the protein concentrations using the Bradford assay (Bio-Rad, Hercules, CA, USA), the resulting supernatants (50 µg of protein) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat milk in TBST for 2 h at room temperature and incubated with rabbit polyclonal anti-p21, anti-p-Rb (1∶1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p16 (1∶1,000; Cell Signaling, Beverly, MA, USA) and rabbit anti-GAPDH (1∶1,000; Sigma, St. Louis, MO, USA) antibodies. After incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody, the protein bands were visualized using an enhanced chemiluminescence system (ECL, Thermo Scientific Pierce, Rockford, IL, USA). ImageJ (NIH) was used to analyze the densities of the bands.
5
Renal Protein Expression Analysis
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Total renal proteins were isolated from renal cortex from each rat and homogenized in lysis buffer (50 mM HEPES ph 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, and complete protease inhibitor (Roche). Protein samples containing 50 μg of total protein were resolved by 8.5% SDS-PAGE electrophoresis and electroblotted onto polyvinylidinedifluoride membranes (Millipore). Membranes were then blocked with 5 % blotting-grade non-fat dry milk. After that membranes were incubated in 0.1 % blotting-grade non-fat dry milk with their respective antibodies. Specific antibodies against α-smooth muscle actin (Sigma A2547, 1:5000), Smad3 (sc-101154, 1:500), phosho-Smad3 (Millipore, 1:500), TGF-β (sc-146, 1:500), ETA (Abcam, 1:5000) and ETB (Abcam, 1:5000) were used. After incubation with primary antibody, membranes were washed and incubated with their respective secondary antibody. As a loading control, membranes were incubated overnight at 4 ºC with goat anti-actin antibody (Santa Cruz Biotechnology, 1:5000 dilution). Actin was detected using donkey anti-goat IgG-HRP (1:5000, Santa Cruz Biotechnology). Proteins were detected with an enhanced chemiluminescence kit (Millipore) and autoradiography, following the manufacturer's recommendations. The bands were scanned for densitometric analysis.
6
Protein Expression Analysis in Cancer
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25μg of protein was loaded and separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). The following antibodies were used to probe the alterations of protein: MACC1 (Sigma, St. Louis, MO),β-catenin, cyclin D1, vimentin, E-cadherin, phosphorylated-GSK3β (phos-GSK3β) (Ser9), MMP9 (Cell Signaling Technology, Danvers, MA),c-Myc, Met, total caspase-3, and cleaved caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA), Signal was detected by enhanced chemoluminescence techniques (Millipore). GAPDH or Histone 3 (Cell Signaling Technology) was used as the loading control.
7
Protein-Protein Interaction Assays
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For the GST pull-down assays, 35S-labeled proteins were produced with the TNT T7-coupled reticulocyte lysate system (Promega, USA), and GST fusion proteins were expressed in Escherichia coli BL21. Radioactively labeled ER or TR proteins were translated in vitro, incubated with various immobilized GST-p53 fusion proteins, and eluted and analyzed using SDS-PAGE as previously described
[14 (link)].
For the co-immunoprecipitation assay, cell extracts containing equivalent amounts of protein were immunoprecipitated in lysis buffer containing the indicated monoclonal antibody against Gal4DBD overnight (4°C). Protein A/G Sepharose beads were added to the immunoprecipitation mixture for 1 hr before three washes with SNNTE buffer. The entire immunoprecipitate was then suspended in SDS-PAGE sample buffer, boiled, and loaded onto an 10% SDS-PAGE. The separated proteins were transferred onto polyvinylidine difluoride membranes (Millipore, USA) and detected using antibodies against HA (3 F10, Hoffmann-La Roche, Switzerland) and Gal4DBD (Santa Cruz Biotechnology, USA).
[14 (link)].
For the co-immunoprecipitation assay, cell extracts containing equivalent amounts of protein were immunoprecipitated in lysis buffer containing the indicated monoclonal antibody against Gal4DBD overnight (4°C). Protein A/G Sepharose beads were added to the immunoprecipitation mixture for 1 hr before three washes with SNNTE buffer. The entire immunoprecipitate was then suspended in SDS-PAGE sample buffer, boiled, and loaded onto an 10% SDS-PAGE. The separated proteins were transferred onto polyvinylidine difluoride membranes (Millipore, USA) and detected using antibodies against HA (3 F10, Hoffmann-La Roche, Switzerland) and Gal4DBD (Santa Cruz Biotechnology, USA).
8
Protein Expression Analysis in Cell Lysates
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Cells were lysed in lysis buffer on ice for 20 minutes. The lysate was centrifuged at 12,000 rpm for 20 minutes at 4 °C. The protein content of the lysate was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equivalent amounts of proteins of cell lysates were boiled with 2× sodium dodecyl sulfate sample buffer for five minutes. Proteins were loaded onto polyacrylamide gels and transferred onto polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked by 1% bovine serum albumin PBS Tween 20 and probed with the following primary antibodies for two hours at room temperature: 1:1,000 matrix metalloproteinase-2 (MMP-2) and 1:1,000 MMP-9 (Cell Signaling Technologies Inc.); 1:11,000 human telomerase reverse transcriptase (hTERT, Santa Cruz Biotechnolgy, Santa Cruz, CA); 1:5,000 glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich Corporation, St Louis, Mo, USA). Blots were probed with secondary antibody-conjugated horseradish peroxidase, and were developed using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Bucks, UK) with enhanced chemiluminescence system film, according to manufacturer’s specifications.
9
Protein Expression Analysis in CRC Cells
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Total protein of CRC cells was prepared using RIPA lysis buffer (Beyotime, Nantong, P.R. China) according to the operating instructions. The protein concentration in the lysates was determined using a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Protein (30 μg) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA). Membranes were then blocked with 5% fat-free milk and incubated with primary antibodies (anti-TRIM37, anti-MMP-2, anti-MMP-9, anti-β-catenin, anti-cyclin D1, anti-c-Myc, and anti-GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After washing three times with TBST, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h. The signals were determined using an enhanced chemiluminescence (Gibco).
10
Western Blot Protocol for Enzyme Analysis
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Cell lysates were prepared in RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with 50 mM NaF, 1 mM Na3VO4, 5 mM EDTA, and 1 mM PMSF, all from Sigma-Aldrich that was freshly added to the lysis solution before each experiment. Similar quantities of lysates were applied to SDS-PAGE gels and transferred to polyvinylidine difluoride membranes (Millipore, Darmstadt, Germany). Membranes were blocked with Tris-buffered saline-Tween 20 0.1% (TBS-t) and 5% non-fat milk (Sigma-Aldrich) for 1 h at room temperature in agitation and incubated overnight with specific antibodies against the CMAH enzyme (Santa Cruz, Dallas, TX), CAIX (Novus Biologicals, Littleton, CO) and GAPDH (Cell Signaling; Danvers, MA). Binding was revealed with a horseradish peroxidase-conjugated anti-rabbit antiserum (Cell Signaling) followed by Luminata Forte (Millipore).
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