To obtain samples for protein electrophoresis, Nicotiana benthamiana leaves were ground to a powder in liquid nitrogen and lysed in a buffer containing three parts of Tris-HCl, pH 7.5 and one part of 4× Laemmli sample buffer (100 m M Tris-HCl pH 6.8, 100 m M β-mercaptoethanol, 10% glycerol, 4% SDS, 0.1% bromophenol blue). The samples were denatured at 95 °C for 5 min and cleared from cell debris via centrifugation. Proteins were separated by 12% polyacrylamide SDS-PAGE and transferred to a Hybond-P polyvinylidene difluoride (PVDF) membrane (GE Healthcare Bio-Sciences, Niskayuna, NY, USA). Rabbit Anti-GFP antibodies conjugated with peroxidase (Rockland, Pottstown, PA, USA) were used for protein detection. After antibody incubation, bands were observed via chemiluminescence using an ECL system (GE Healthcare Bio-Sciences).
Hybond p polyvinylidene difluoride pvdf membrane
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Hybond-P is a polyvinylidene difluoride (PVDF) membrane manufactured by GE Healthcare for use in laboratory applications. The membrane is designed for protein transfer and immobilization in western blotting, dot blotting, and other protein-based assays. It provides a stable and durable platform for the detection and analysis of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin, by Chrono-Log (2 mentions)
Ecl system, by GE Healthcare (1 mentions)
Hinokitiol, by Merck Group (1 mentions)
Anti phospho pi3k, by Cell Signaling Technology (1 mentions)
Recombinant pdgf bb, by Thermo Fisher Scientific (1 mentions)
Anti p53, by GeneTex (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Anti rorγt, by Abcam (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Mini protean electrophoresis system, by Bio-Rad (1 mentions)
Sodium fluorescein, by Merck Group (1 mentions)
Luciferin luciferase, by Merck Group (1 mentions)
Phorbol 12 13 dibutyrate pdbu, by Merck Group (1 mentions)
Anti p selectin, by BioLegend (1 mentions)
Horseradish peroxidase hrp conjugated affinipure goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
U46619, by Chrono-Log (1 mentions)
Stains all, by Merck Group (1 mentions)
Anti lubricin, by Abcam (1 mentions)
10 protocols using hybond p polyvinylidene difluoride pvdf membrane
1
Extraction and Detection of GFP-tagged Proteins
2
Signaling Pathways Modulation by Hinokitiol
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Hinokitiol (product number: 469521; purity: ≥ 98.5%) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Recombinant PDGF-BB was purchased from Pepro-Tech (Rocky Hill, NJ, USA). Anti-mouse and anti-rabbit immunoglobulin G-conjugated horseradish peroxidase (HRP) was purchased from GE Healthcare (Sunnyvale, CA, USA) and/or Jackson-Immuno Research (West Grove, PA, USA). Anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-AKT (Ser473), antiphospho-PI3K, and antiphospho-JAK2 monoclonal antibodies (mAbs) were purchased from Cell Signaling (Beverly, MA, USA). Anti-p53 was obtained from GeneTex Inc (Irvine, CA). The WWP-luc (p21cip/Waf1 promoter) construct (Addgene plasmid 16451) and the PG13-luc plasmid with p53 binding sites (Addgene plasmid 16642) were kindly provided by Dr. Ming Jen Hsu. The Dual-Glo luciferase assay system was purchased from Promega (Madison, WI). The Hybond-P polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system were obtained from GE Healthcare (Sunnyvale, CA, USA). All other chemicals used in this study were of reagent grade.
3
Immunoblotting of Epsilon Prototoxin
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SDS-PAGE was performed with 12% polyacrylamide slab gels using the Laemmli protocol. Dilutions of epsilon prototoxin in 60 mM Tris-HCl pH 6.8, 2.5% β-mercaptoethanol, 2% SDS, 10% glycerol and 0.01% bromophenol blue were boiled for 10 min before migration. After electrophoresis, the antigens were transferred to Hybond-P polyvinylidene difluoride (PVDF) membrane (GE Healthcare). The PVDF sheets were processed with the SNAP-ID™ western blot instrument (Millipore), blocked with 0.25% milk in PBS containing 0.1% Tween 20 and probed with 4 μg/mL of each purified mAb in blocking solution for 10 min. After reaction for 10 min with peroxidase-conjugated goat anti-mouse IgG antibody in blocking solution, the blot was further stained by chemiluminescence (ECL Prime, GE Healthcare).
4
Quantitative Analysis of Th17 Markers
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Protein expression of IL-26, IL-17A and RORγt were determined by Western blotting. SDS-PAGE was performed on 12.5% gels using a Mini-Protean Electrophoresis system (Bio-Rad, Switzerland) and blotted onto a hybond-P polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Glattbrugg, Switzerland) in the same device. Membranes were blocked with 5% milk powder in TBS-T (50 mM Tris, Calbiochem, pH 7.6; 150 mM NaCl, 0.1% Tween-20; Sigma-Aldrich) and incubated overnight at 4°C with mouse anti-bodies, anti-IL-26 (KU32-52; BioLegend), anti-1L17A (Abcam), anti-RORγt (Abcam) or β-Actin antibodies (Sigma) in 10% FCS in TBS-T. HRP-conjugated secondary antibodies (anti-mouse; Cell Signaling, Danvers, MA, USA) were used. The ECL chemiluminescence reagent was used to detect the signal bands as and semi-quantitative analyses using densitometry were performed using Image J version 1.48v (National Institutes of Health, Bethesda, MD).
5
Platelet Signaling Pathway Analysis
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Thrombin, U46619, collagen and convulxin were purchased from Chrono‐Log. Fluorescein sodium, phorbol 12,13‐dibutyrate (PDBu), luciferase/luciferin and ADP were purchased from Sigma. Fura 2‐AM; anti‐phospho‐Lyn (Tyr507), anti‐phospho‐Akt (Ser473), anti‐phospho‐Fyn (Tyr530) and anti‐Lyn polyclonal antibodies (pAbs); and anti‐Fyn monoclonal antibodies (mAbs) were purchased from Abcam. Anti‐pleckstrin (p47) and anti‐phospho‐ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) pAbs were purchased from GeneTex. Anti‐phospho‐(Ser) PKC substrate, anti‐phospho‐JNK (Thr183/Tyr185), anti‐phospho‐p38 MAPK (Thr180/Tyr182) and anti‐JNK pAbs; and anti‐Akt, anti‐p38 MAPK and anti‐ERK mAbs were purchased from Cell Signaling. Horseradish peroxidase (HRP)‐conjugated AffiniPure goat antirabbit, AffiniPure goat antimouse and AffiniPure donkey antigoat immunoglobulin G (IgG) were purchased from Jackson ImmunoResearch. Allophycocyanin (APC)‐conjugated PAC‐1 antibodies and anti‐P‐selectin were purchased from Biolegend. Hybond‐P polyvinylidene difluoride (PVDF) membrane was purchased from GE Healthcare Life Sciences. A SuperLight Chemiluminescent HRP kit was purchased from Bionovas.
6
Lubrication Testing Materials Protocol
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Reagents for lubrication testing materials were as described previously,2 (link),6 (link),33 (link) including HA in a 4000-kDa form (Healon; Advanced Medical Optics). The antibody to PRG4 was anti-Lubricin from AbCam,2 (link) nonspecific rabbit IgG was from Pierce, and mouse anti-rabbit IgG secondary antibody was from Jackson ImmunoResearch. Streptomyceshyaluronidase was from Seikagaku, SeaKem gold agarose was from Lonza; 50X TAE (2M Tris, 0.5 M ethylenediamine tetraacetic acid [EDTA]) electrophoresis buffer was from Life Technologies, Hybond-P polyvinylidene difluoride (PVDF) membrane for Western blotting was from GE Healthcare, and Stains-All was from Sigma-Aldrich.
7
Western Blot Analysis of Antibody Responses
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Resolved proteins were transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK) using a Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) according to the manufacturer's instructions. The membranes were subsequently blocked using 5% non-fat milk in 20 mM Tris containing 500 mM NaCl (Tris-buffered saline) and 1% Tween 20 (TBS-T) for 1 h at room temperature. The blocking solution was then discarded and the membranes incubated overnight at 4°C with a 1∶200 dilution of immune sera collected on day 14 post-infection from mice immunized with CW and CP protein preparations. The membranes were then washed six times in TBS-T and antibody binding detected by the addition of goat anti-mouse IgG HRP-conjugated antibody (Pierce Biotechnology Inc., Rockford, IL) diluted 1∶1000 in TBS-T containing 5% non-fat milk for 1 h at room temperature. After six washes in TBS-T, the membranes were briefly incubated with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology Inc.) and protein spots detected using a ChemiDoc XRS Camera and Quantity One 1-D analysis software (Bio-Rad).
8
Western Blot Analysis of MAPK Phosphorylation
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Microglial cell lysates were boiled after the addition of sample buffer (1 M Tris-HCl, 20% sodium dodecyl sulfate (SDS), and 2.5% glycerol). Fifty micrograms of total protein were separated on a 5 to 20% Tris-glycine SDS-polyacrylamide gel and blotted onto Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare UK, Buckinghamshire, UK). Membranes were blocked with 1% skim milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature. Primary antibodies to detect phosphorylated and total MAPK (Cell Signaling, Danvers, MA, USA) were applied at the concentrations recommended by the manufacturers. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare), which was used at a dilution of 1:1000. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL, USA) was used according to the manufacturer’s instructions. The intensities of the bands were calculated using the CS Analyzer 1.0 (Atto Corporation, Tokyo, Japan).
9
Collagen and Thrombogenic Pathway Activation
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Collagen (type I), 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin (U46619), and thrombin were purchased from Chrono-Log Corporation (Havertown, PA, USA). Anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), anti-phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185), anti-phospho-(Ser) protein kinase C (PKC) substrate, anti-JNK polyclonal antibodies (pAb), and anti-p38 MAPK and anti-Akt monoclonal antibodies (mAb) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-Akt (Ser473) pAb was purchased from Biorbyt (Cambridge, UK), and anti-pleckstrin (p47) pAb was purchased from Gene Tex (Irvine, CA, USA). Hybond-P polyvinylidene difluoride (PVDF) membranes, enhanced chemiluminescence (ECL) Western blotting detection reagent, and the analysis system were purchased from GE Healthcare Life Sciences (Buckinghamshire, UK). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
10
Protein Extraction and Western Blot Analysis
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Protein extraction and western blot analysis were performed as previously described2 (link). The A. nidulans cultures were ground using liquid nitrogen, and cells were resuspended in protein extraction buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, and 1% NP-40) with 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium fluoride, and 1 mM sodium vanadate. The supernatant was obtained after centrifugation at 15,000 × g at 4 °C for 30 min. Total protein samples were electrophoresed on 8% SDS-PAGE and subsequently electroblotted onto Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare). The membrane was blocked with 5% skimmed milk, and protein detection was carried out using anti-FLAG (Sigma-Aldrich) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) secondary antibody following the manufacturers’ protocols (ELPISBIO). SDS-PAGE and silver staining kit (ELPISBIO) were used for silver staining.
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