EC9706 cells were solubilized in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) with protease inhibitors (Roche, Basel, Switzerland). The protein concentrations were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA) according to the manufacturer’s instructions. Proteins (50 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific, Inc.). The membranes were then incubated with 5% milk–Tris-buffered saline-Tween (TBST) blocking buffer for 3 h at room temperature. After washing three times with PBS, the membranes were incubated with primary antibodies of B-cell lymphoma 2 (Bcl-2; ab59348), Bcl-2-associated X (Bax; ab53154), caspase 3 (ab32531), caspase 9 (ab32539), CXCR4 (ab124824), Wnt3a (ab28472), Wnt5a (ab72583), β-catenin (ab32572), phosphorylated inhibitor of NF-κB (p-IκBα; ab92700), total (t)-IκBα (ab32518), p-p65 (ab86299), t-p65 (ab32536), and GAPDH (ab181602; Abcam, Cambridge, UK) at 4°C overnight, subsequently added horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205718; 1:5,000; Abcam), and incubated at room temperature for 1 h. An enhanced chemiluminescent kit (Thermo Fisher Scientific, Inc.) was then used to conduct chemiluminescent detection.
Polyvinylidene fluoride pvdf membrane
Manufactured by Thermo Fisher Scientific
Sourced in United States
Polyvinylidene Fluoride (PVDF) membranes are a type of laboratory equipment used for a variety of applications. PVDF membranes are known for their chemical and thermal resistance, as well as their ability to withstand organic solvents. They are commonly used in Western blotting, dot blotting, and protein and nucleic acid transfer applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Ab53154, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Enhanced chemiluminescent kit, by Thermo Fisher Scientific (1 mentions)
Ab32539, by Abcam (1 mentions)
Ab124824, by Abcam (1 mentions)
Ab92700, by Abcam (1 mentions)
Ab32531, by Abcam (1 mentions)
Ab28472, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab32536, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
1 step tmb blotting substrate solution, by Thermo Fisher Scientific (1 mentions)
Nativepage 3 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
15 protocols using polyvinylidene fluoride pvdf membrane
1
Protein Expression in EC9706 Cells
2
Western Blot Analysis of Apoptosis Markers
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After the treatments with Tacotanina and PTX for 16, 24, and 48 h, the MDA-MB-231 cells were collected. Lysis buffer (20 mM Tris HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1.0% NP-40, and 10 mM EDTA) was used to obtain a total protein extract of each sample. To measure the concentration of the total protein the bicinchoninic acid (BCA) assay (Pierce) was used. In this case, 30 μg of protein was loaded in 11% SDS-PAGE. Proteins were blotted using polyvinylidene fluoride (PVDF) membranes (Thermo-Fisher Scientific, Waltham, MA, USA), subsequently blocked for 1 h with 5.0% (w/v) BSA/TTBS and incubated overnight at 4 °C with the primary antibodies: anti-Bcl-2 (N1N2) (GeneTex, Irvine, California, USA), anti-cleaved-casp-3 (Thermo Fisher Scientific, Waltham, MA, USA), anti-XIAP (D2Z8W), anti-Bax (D2E11) anti-COX IV (3E11) (Cell signaling) the latter was used as the loading control. The next day, the membranes were incubated for 1 h at room temperature, with the corresponding secondary antibody, either anti-mouse IgG or anti-rabbit IgG (Merck Group, DE, Darmstadt, Germany) [35 (link)]. Bands detection was performed by using the chromogenic HRP system 1-Step™ TMB-Blotting Substrate Solution (Thermo-Fisher Scientific, Waltham, MA, USA) yields a blue-colored precipitate. ImageJ software was used to normalize and semi-quantify bands’ density.
3
SDS-PAGE and BN-PAGE Analysis of Cytb6f Protein
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For SDS–PAGE analysis of purified cytb6f, protein samples were mixed with an equal volume of 2× Laemmli sample buffer (Merck) and boiled for 10 min prior to separation on precast NuPAGE 12% Bis-Tris gels (Invitrogen). For BN-PAGE analysis, cytb6f was diluted in 4× sample buffer (100 mM Tris–HCl pH 7.5, 0.05% (w/v) bromphenol blue, 40% (w/v) glycerol) and analyzed on precast NativePAGE 3–12% Bis-Tris gels (Invitrogen). Gels were stained with Coomassie Brilliant Blue and imaged using an Amersham 600 imager (GE Healthcare). Alternatively, SDS–PAGE separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher Scientific) as described previously [37 (link)] and cytochrome c-mediated chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) and an Amersham Imager 600 (GE Healthcare).
4
Protein Expression Analysis by Western Blot
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Protein extraction was conducted by Radioimmunoprecipitation assay (RIPA) Lysis Buffer for tissues and cells (Solarbio). Protein products were electrophoresed on sodium dodecyl sulfate polyacrylamide gel (Invitrogen) and transferred onto the Polyvinylidene Fluoride (PVDF) membranes (Invitrogen), then the membranes were soaked in 5% nonfat dry milk (Cell Signaling Technology, CST; Boston, MA, USA) for protein blocking. After the incubation of primary antibodies at 4°C overnight and secondary antibody (CST, #7074, 1:3000) at room temperature for 1 h, the protein blots were discerned using ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) and the expression analysis was performed by Image Lab software (Bio-Rad). Primary antibodies used in this study were listed as follows: E-cadherin (CST, #3195, 1:1000), Vimentin (CST, #5741, 1:1000), Snail (CST, #3879, 1:1000), HMGA1 (CST, #7777, 1:1000), β-actin (CST, #4970, 1:1000).
5
Indoamine Oxidase Pathway Modulation
6
Analyzing KRAS Protein Expression in Panc-1 Cells
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Panc-1 cells in the logarithmic growth phase were first collected and digested with 0.25% trypsin. Next, radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Shanghai, China) was applied to extract total protein. A bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) was employed for the quantification of protein concentrations and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad) was utilized for protein separation. After that, polyvinylidene fluoride (PVDF) membranes (Invitrogen) were employed for protein transference following the producer’s guidelines and sealed in 5% skim milk at 37°C for 2 h. The membranes were placed into a plastic bag in the next step and incubated overnight with primary antibodies against KRAS (ab180772, 1 : 500, Abcam, Cambridge, MA, USA) and GAPDH antibody (ab9485, 1 : 2000, Abcam) at 4°C. After 3 times 5-min washing with phosphate buffered saline (PBS), at room temperature, 1.5-h incubation of membranes with secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (1 : 2000) was conducted, followed by again washing 3 times for 5 min with PBS. An electrochemiluminescent (ECL) detection system (Thermo Scientific, MA, USA) was employed for signal detection. The density of protein bands was quantified or analyzed using Quantity One v4.6.2 software (Bio-Rad).
7
Western Blot Analysis of Apoptosis and Wnt Signaling
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This method was used to semi-quantify the proteins as mentioned below, and cells were harvested and lysed by RIPA Lysis Buffer (Beyotime) to obtain total protein. After that, 40 μg of total protein was measured with BCA protein Assay Kit (Beyotime). Thereafter, proteins were isolated with 8% SDS-PAGE and transferred into Polyvinylidene Fluoride (PVDF) membranes (Invitrogen) later. Next, 8% skimmed milk powder was selected to block membranes for 2 hours and anti-pro-caspase-3 (1:1000; ab183179, Abcam), anti-cleaved-caspase-3 (1:1000; ab49822), anti-p53 (1:1000; ab26), anti-PARP1 (1:1000; ab227224), anti-Wnt3a (1:1000; ab219412), anti-Wnt5a (1:1000; ab227229), anti-β-catenin (1:1000; ab32572) and anti-GAPDH (1:2000; ab181602) were incubated with PVDF membranes at 4°C overnight. Afterwards, membranes were rinsed by Pierce™ Protein-Free T20 (PBS) Blocking Buffer (Thermo Scientific) three times and incubated with secondary antibodies -Goat Anti-Rat IgG H&L (HRP) (ab97057) at room temperature for 2 hours. Finally, BeyoECL Moon (Beyotime) was used for testing protein.
8
Nicotinic Receptor Signaling Pathway Protocol
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(-)-Nicotine hydrogen tartrate, scopolamine hydrobromide, (2.4)-dimethoxybenzylidene anabaseine dihydrochloride (DMXBA, also called GTS-21), sodium orthovanadate, protease inhibitor cocktail, Tween 20, Tween 80, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (NaCl) was purchased from Baxter (Lublin, Poland). Methyllycaconitine (MLA), and antibodies for the α7 nAChR (#ab23832) and β-actin were obtained from Abcam (Cambridge, UK). Antibodies detecting phospho-ERK1/2 (#4376, Thr202/Tyr204) and total ERK1/2 (#4695) were purchased from Cell Signaling Technology (Danvers, MA, USA). EDTA and EGTA were purchased from Boston BioProducts (Ashland, MA, USA). Phenylmethanesulfonyl fluoride (PMSF) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Phosphatase inhibitor cocktail sets I and II were purchased from Calbiochem (San Diego, CA, USA). The bicinchoninic acid assay kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene fluoride (PVDF) membranes and 4-12% pre cast polyacrylamide gels were purchased from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the Cell Lysis Buffer was obtained from Cell Signaling Technology (MA, USA). PAM-2 was synthesized as described in Arias et al [8] .
9
Fluorescent Tau13 Antibody Western Blot
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For western blots, a fluorescent anti-tau antibody was used. Therefore tau13 (Biolegend, San Diego, USA) was labelled with CF633 (Biotium, Freemont, USA), and the labelling process was performed as described previously [37 (link)]. 2N4R tau recombinant protein samples were prepared in Laemmli buffer (final 1× composition: 20 mM Tris, pH 6.8, 2% SDS, 6% glycerol, 1% β-ME, 0.002% Bromophenol Blue). All samples were heated at 95°C for 5 min and separated using SDS PAGE (15%). Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, USA) at 500 mA for 40 min. After a washing step for 15 min in Tris-buffered saline tween buffer (TBS-T) (20 mM Tris, 150 mM NaCl, 0.1% Tween 20), the membrane was blocked for 1 h with 2.5% milk powder/TBS-T. Next, the membrane was washed with TBS-T, 2 × 5 min and in the last step for 15 min. tau13 stocks were 1 mg/ml and were diluted in TBS-T (1:5000). The membrane was incubated with the antibody for 1.5 h. at RT. After a final wash step (2 × 5 min and 1 × 10 min), TBS-T was performed. Detection based on the CF633 fluorescence of the labelled tau13 antibody. Bio-Rad universal hood II and Chemidoc XRS camera and Quantity One 4.6.5 software enabled the visualisation and quantification of the protein bands.
10
Western Blot Analysis of Protein Expression
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Cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8], 1% Triton X, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) with the addition of protease and phosphatase inhibitor tablets (Pierce). Protein concentration was quantified using the BCA protein assay kit (Pierce), with 50 μg of protein per sample loaded onto gels. Proteins were separated on 7.5% or 10% SDS-PAGE gels, transferred onto polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific), and probed for CTR1 (sc-66847) used at 1:1,000, ABCB9 (sc-393412) used at 1:1,000, LAMP1 (sc-17768) used at 1:1,000, OCT1 (sc-293181) used at 1:1,000, β-actin (sc-130300) used at 1:10,000 (Santa Cruz Biotechnology), and γH2A.X (9718) used at 1:1,000 (Cell Signal), followed by incubation with anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (P0260) used at 1:2,000 (Dako) or anti-rabbit HRP-conjugated secondary antibody (sc-2004) used at 1:2,000 (Santa Cruz Biotechnology). Bands were detected using Clarity western enhanced chemiluminescence (ECL) substrate (Bio-Rad) and visualized using the ChemiDoc imaging system (Molecular Imager ChemiDoc XRS with Image Lab 3.0). Densitometric analysis was performed with Image Lab 3.0 software (Bio-Rad).
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