For protein analyses the cells from different blood donors were pooled to get sufficient amounts of proteins, and whole cell lysates were prepared in the passive lysis buffer of Dual Luciferase Assay Kit (Promega) containing 10 mM Na3PO4. Equal amounts of proteins (10–30 ug/lane) were separated on SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Antibodies against IRF3 and MxA were as previously described21 (link),22 (link) and antibodies against ZIKV NS5 were prepared as described above. The staining was done in blocking buffer at RT for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3, #4947), STAT2 (#72604), P-STAT2 (#88410) and GAPDH (#2118) were from Cell Signaling Technology, for β-Actin (SC-10731) from SantaCruz Biotechnology, and the stainings were done in Tris-buffered saline, pH 7.4 containing 5% BSA at +4 °C overnight. HRP-conjugated antibodies (Dako) were used in the secondary staining at RT for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).
Hybond p polyvinylidene difluoride pvdf membrane
Manufactured by Cytiva
Sourced in United Kingdom
Hybond-P is a polyvinylidene difluoride (PVDF) membrane commonly used for protein transfer and detection in Western blotting applications. It provides a stable and reliable platform for protein immobilization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus system, by GE Healthcare (5 mentions)
Dual luciferase assay kit, by Promega (5 mentions)
Sheep anti mouse igg, by Cytiva (5 mentions)
Horseradish peroxidase hrp conjugated donkey anti rabbit immunoglobulin g igg, by Cytiva (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hrp conjugated antibodies, by Agilent Technologies (3 mentions)
Enhanced chemiluminescence ecl western blotting detection reagent, by Cytiva (3 mentions)
Horseradish peroxidase hrp conjugated antibody, by Agilent Technologies (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh 2118, by Cell Signaling Technology (2 mentions)
Hypermax films, by GE Healthcare (2 mentions)
Phorbol 12 13 dibutyrate pdbu, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti mouse and anti rabbit immunoglobulin g conjugated horseradish peroxidase hrp, by Cytiva (2 mentions)
Thrombin, by Merck Group (2 mentions)
Enhanced chemiluminescence western blotting detection reagent, by Cytiva (2 mentions)
Collagen type 1, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
P stat2, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
18 protocols using hybond p polyvinylidene difluoride pvdf membrane
1
Protein Analysis via Western Blotting
2
Immunoblotting Analysis of Lung Proteins
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Homogenized lungs were lysed on ice in 500 μl lysis buffer [20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2 mM EDTA (pH 7.4), 1% Triton X-100, 10% glycerol, 1 mM sodium vanadate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 25 mM glycerophosphate and 10 μg/ml leupeptin]. Lysates were cleared by centrifugation and the protein concentration was determined. SDS-PAGE gel electrophoresis of the cell lysates were performed as described [29 (link)]. After electrophoresis, proteins were transferred to pretreated Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were incubated for 1h at room temperature in blocking buffer (5 % non-fat dry milk in PBS containing 0.1% Tween) and subsequently for 12 h at 4°C in blocking buffer with primary antibodies raised against β-actin (1/5000, Sigma) or FGF2 (clone FB-8, 1/5000, Sigma). After washing, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (anti-mouse: 1/2000 or anti-rabbit: 1/4000, Dako) in blocking buffer for 25 min at room temperature. Next, the membranes were washed extensively. Immunoreactive proteins were detected by chemiluminescence (ECLplus, Bio-Rad). Samples were collected from 3 independent experiments.
3
Western Blot Analysis of Protein Expression
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For protein expression analyses, cells from different blood donors were pooled to obtain sufficient amounts of protein. The whole-cell lysates from cell lines or pooled primary cells were prepared in the passive lysis buffer of the dual luciferase assay kit (Promega) containing 10 mM Na3PO4. Equal amounts of protein (10 to 30 μg/lane) were separated by SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Antibodies against IRF3 and MxA were as previously described (74 (link), 75 (link)), and antibodies against SARS-CoV N protein and SARS-CoV-2 S1 protein were prepared as described above. Staining was done in blocking buffer at room temperature for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3; 4947), p38 (9212), phosphorylated p38 (P-p38; 9211L), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 2118) were from Cell Signaling Technology, and staining was done in Tris-buffered saline, pH 7.4, containing 5% bovine serum albumin (BSA) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated antibodies (Dako) were used in the secondary staining at room temperature for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).
4
Protein Expression Analysis of Immune Cells
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For protein expression analyses the cells from different blood donors were pooled to obtain sufficient amounts of protein. The whole cell lysates from pooled primary immune cells or A549 cells were prepared in the passive lysis buffer of Dual Luciferase Assay Kit (Promega) containing 10 mM Na3VO4. Equal amounts of proteins (10–30 µg/lane) were separated on SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Rabbit antibodies against IRF3 and MxA [37] (link), [38] (link) and SARS-CoV-2 S1 protein [19] have been previously described. To obtain adenovirus hexon-specific antibodies one rabbit was immunized with a human dose of AZD1222 for three times at 3 week intervals followed by the collection of immune serum 10 days after the last dose. All antibody stainings were done in blocking buffer at RT for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3, #4947), and GAPDH (#2118) were from Cell Signaling Technology and staining was done in Tris-buffered saline, pH 7.4 containing 5% BSA at + 4 °C overnight. HRP-conjugated antibodies (Dako) were used in the secondary staining at RT for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).
5
Western Blot Analysis of Protein Expression
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Western blot was conducted as described previously (16 (link)). Briefly, cells were lysed and quantified using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Shanghai, China). Protein lysates (15 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Uppsala, Sweden). After blocking with 5% non-fat dry milk in a tris-buffered saline buffer for 2 h at room temperature, the blots were incubated at room temperature first with diluted antibodies against various proteins (MAPK15, FYN, IKBKB, MAP2K6, CDK4, dilution 1:1000; γ-H2AX, 1:5000) for 2 h and then with horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (Abcam, Cambridge, USA) for 1 h. The signals were visualized with an enhanced chemiluminescence detection reagent (Abcam, Cambridge, USA). β-actin (1:5000) served as a loading control.
6
Western Blot Analysis of Protein Expression
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For protein expression analyses, cells from different blood donors were pooled to obtain sufficient amounts of protein. The whole-cell lysates from cell lines or pooled primary cells were prepared in the passive lysis buffer of the dual luciferase assay kit (Promega) containing 10 mM Na3PO4. Equal amounts of protein (10 to 30 μg/lane) were separated by SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Antibodies against IRF3 and MxA were as previously described (74 (link), 75 (link)), and antibodies against SARS-CoV N protein and SARS-CoV-2 S1 protein were prepared as described above. Staining was done in blocking buffer at room temperature for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3; 4947), p38 (9212), phosphorylated p38 (P-p38; 9211L), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 2118) were from Cell Signaling Technology, and staining was done in Tris-buffered saline, pH 7.4, containing 5% bovine serum albumin (BSA) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated antibodies (Dako) were used in the secondary staining at room temperature for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).
7
Protein Analysis of Blood Samples
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For protein analyses the cells from different blood donors were pooled, and whole cell lysates were prepared in passive lysis buffer of Dual Luciferase Assay Kit (Promega) containing 1 mM Na3VO4. Total cellular proteins were denatured in the Laemmli buffer and boiled before transferring the samples out from the BSL-3 facilities. Equal proportion of samples were separated on SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS (blocking buffer). The rabbit antibodies against IRF1, IRF3, IRF7, MxA, influenza A NP and M1 were prepared as described previously [19] (link), [29] (link)–[31] (link). The staining was done in blocking buffer at RT for 1 h. Antibodies for phosphorylated IRF3 (P-IRF3), IκBα, and GAPDH were from Cell Signaling Technology, and the staining was done in PBS containing 5% BSA at +4°C overnight. Antibodies against IFITM3 were from Abgent. HRP-conjugated antibodies (Dako) were used in the secondary staining at RT for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).
8
Western Blot Quantification Protocol
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Cells were lysed in Passive Lysis Buffer (Promega) supplemented with 1 mM Na3VO4. Total protein concentrations were determined using the Bio-Rad protein assay. SDS-PAGE was used to separate equal amounts of protein (10 µg -20 µg) which was followed by transfer of the proteins onto Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). For in-house antibodies, membranes were blocked for 30 min at room temperature (RT) in blocking buffer (5% fat-free milk in PBS with 0.05% Tween-20). Primary and secondary antibodies were diluted in blocking buffer and membranes were incubated for 1h at RT. All washes were carried out for 3 x 10 min in PBS with 0.05% Tween-20. For commercial antibodies blocking and staining were performed according to manufacturer’s instructions. Protein bands were visualised using Pierce™ ECL Western Blotting Substrate (Thermo Fisher) and BIOMAX XAR films (CareStream). Quantification of immunoblots was carried out using ImageJ software (31 (link)).
9
Collagen Signaling Pathway Activation
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Type I collagen and phorbol-12, 13-dibutyrate (PDBu) were purchased from Sigma (St Louis, MO). Fura 2-AM was purchased from Molecular Probe (Eugene, OR). The anti-Akt (pan) (40D4) monoclonal antibody (mAb), anti-phospho-Akt (Ser473) polyclonal antibody (pAb), anti-phospho-(Ser) protein kinase C (PKC) substrate pAb, anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) pAb, anti-p38 MAPK (5F11) mAb, anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) pAb, anti-p44/42 MAPK (137F5) mAb, anti-phospho-c-Jun N-terminal kinse (JNK) (Thr183/Tyr185) mAb, and anti-JNK pAb were purchased from Cell Signaling (Beverly, MA). The anti-α-tubulin mouse mAb was purchased from Thermo Scientific (Waltham, MA). The Hybond-P polyvinylidene difluoride (PVDF) membrane, an enhanced chemiluminescence (ECL) western blotting detection reagent, a horseradish-peroxidase (HRP)-conjugated donkey anti-rabbit IgG, and a sheep anti-mouse IgG were purchased from Amersham (Buckinghamshire, UK).
10
Andrographolide Modulates NF-κB Signaling
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Dulbecco's modified Eagle's medium (DMEM), trypsin (0.25%), L-glutamine, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA). Andrographolide (≥98%), TNF-α, LY294002, SP600125, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The anti-iNOS rabbit polyclonal antibody (pAb) and the anti-p65 antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); the anti-α-tubulin mouse monoclonal antibody (mAb) was purchased from Thermo Scientific (Waltham, MA, USA); and the anti-phospho-p38 MAPK Thr180/Tyr182 rabbit pAb, anti-p38 MAPK, anti-phospho-p44/p42 extracellular signal-regulated kinase (ERK1/2) Thr202/Tyr204 rabbit pAb, anti-ERK1/2 antibody, anti-phospho-JNK Thr183/Tyr185 rabbit mAb, anti-JNK antibody, anti-phospho-Akt Ser473 rabbit pAb, anti-Akt antibody, anti-phospho-p65 Ser536 rabbit pAb, and anti-IκBα antibody were purchased from Cell Signaling (Danvers, MA, USA). A hybond-P polyvinylidene difluoride (PVDF) membrane, an enhanced chemiluminescence (ECL) western blotting detection reagent and analysis system, the horseradish-peroxidase- (HRP-) conjugated donkey anti-rabbit immunoglobulin G (IgG), and the sheep anti-mouse IgG were acquired from Amersham (Buckinghamshire, UK). Andrographolide was dissolved in 0.1% DMSO and stored at 4°C until it was used.
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