Treated rat NP cells were immediately placed on ice and washed with cold PBS. To prepare the total cellular proteins, the cells were lysed with lysis buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, complete protease inhibitor cocktail (Roche, IN, USA), 1 mM NaF, and 1 mM Na3VO4. Heat-denatured samples were separated on sodium dodecyl sulfate polyacrylamide gels and electrotransferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were then blocked with blocking buffer (5% BSA and 0.1% NaN3 in PBS) and subsequently incubated overnight at 4 °C with anti-CCN2 (1:1000, Santa Cruz Biotechnology or Abcam, Cambridge, UK) antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Tokyo, Japan). Chemiluminescent signals were visualized with an Immobilon Western Chemiluminescent HRP Substrate (Millipore) and scanned using an Ez-Capture MG imaging system (ATTO, Tokyo, Japan). The western blot data were quantified using Image J pixel analysis (NIH Image software). Western blot data are presented as band intensity normalized to that of the loading control (β-actin). To measure the band intensity, the data shown are representative of at least three independent experiments.
Immobilon p polyvinylidene difluoride membrane
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Immobilon-P polyvinylidene difluoride (PVDF) membranes are a type of lab equipment used for protein and nucleic acid transfer and immobilization. They provide a stable, hydrophobic surface for the binding and retention of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hrp substrate, by Merck Group (16 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (9 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (5 mentions)
Pierce western blot ecl plus, by Thermo Fisher Scientific (4 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Image studio lite, by LI COR (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Enhanced hrp dab chromogenic substrate kit, by Tiangen Biotech (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Complete protease inhibitor cocktail, by Merck Group (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Anti flag m2 affinity gel, by Merck Group (3 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (3 mentions)
Westernbright ecl, by Advansta (3 mentions)
Ponceau s staining, by Merck Group (3 mentions)
Complete edta free protease inhibitor cocktail, by Roche (3 mentions)
132 protocols using immobilon p polyvinylidene difluoride membrane
1
Protein Extraction and Western Blot Analysis
2
Quantitative Western Blot Analysis of LasB
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Western blot analyses for LasB were performed using standard protocols as previously described [37 (link)]. Briefly, equal concentration of protein samples (from bacterial culture supernatants) were resolved by SDS-PAGE and electro-blotted onto Immobilon P polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were then incubated for 60 min at room temperature in blocking solution (3% bovine serum albumin in PBS), followed by a 4-h incubation with polyclonal antibody against P. aeruginosa LasB (a gift from Professor Dennis Ohman, Virginia Commonwealth University). The membranes were hybridized with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody. The immune complexes were visualized using the Amersham ECL Western Blotting Detection System (Danaher Corporation, Washington, D.C., USA) on HyBlot CL autoradiography films (Denville Scientific, InC, Metuchen, NJ, USA). In order to assure reproducibility, western blotting was performed twice with similar results. Protein expression levels were quantified by densitometry normalized to the number of bacteria within each sample by using the ImageJ software.
3
Western Blot Protein Detection Protocol
4
Western Blot Analysis of PDX Samples
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Cells and PDX organoids (PDX-O) were lysed with RIPA buffer (Pierce) supplemented with protease inhibitor cocktail (Roche) and PhosStop phosphatase inhibitor mix (Roche). PDX lysate was obtained by mechanical homogenization, reconstituted in RIPA buffer. Protein extracts (20 μg) were separated on 4%–12% NuPAGE Bis-Tris gel (Invitrogen) by electrophoresis and subsequently transferred onto Immobilon-P polyvinylidene difluoride membranes of 0.45 μm pore size (Millipore). Chemiluminescence was detected on the Chemidoc Touch imaging system (Bio-Rad). Detailed antibodies in Supplementary Table S3.
5
Immunoblotting of GFP and HA-tagged Atg8
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Whole cell lysates were separated by SDS-PAGE using 10% (for GFP-TbAtg8) or 12% (for HA-TbAtg8) acrylamide gels. For immunoblotting, proteins were transferred onto Immobilon P polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) by semi-dry blotting for 75 min at 2.5 mA/cm2. Mouse monoclonal antibodies against GFP (Roche Diagnostics, Rotkreuz, Switzerland) or HA (Covance) were used at a dilution of 1∶3000. Mouse monoclonal antibody against eukaryotic elongation factor 1A (eEF1A; Upstate, Lake Placid, NY) was used at a dilution of 1∶5000. Primary antibodies were detected using rabbit anti-mouse IgG conjugated to horseradish peroxidase (Dako, Baar, Switzerland), at a dilution of 1∶5000, by enhanced chemiluminescence (Pierce, Lausanne, Switzerland).
6
Western Blot Analysis of Protein Complexes
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SDS-PAGE loading buffer was added to cleared whole-cell lysates or to samples containing purified protein complexes and resolved on 10% SDS-PAGE gels (Criterion Tris-HCl; Bio-Rad). For Western analysis, resolved proteins were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore) and probed using the antibodies described in Table S2 . Generally, goat anti-mouse IgG–horseradish peroxidase (HRP) secondary antibodies were used, except where mouse anti-V5 primary antibodies were used to probe Western blots of anti-KREPA2-immunoprecipitated proteins. Here, protein A-conjugated HRP was employed to prevent detection of denatured anti-KREPA2 IgG heavy chain. Blots were developed with an enhanced chemiluminescence kit (Thermo Scientific) per the manufacturer’s instructions and imaged using the FluorChem E system (ProteinSimple) or X-ray film (Kodak).
7
Western Blot Analysis of Protein Samples
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Cells were lysed with lysis buffer containing 1% Triton X-100, and the resulting samples separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). Immunoblot analysis was performed and visualized using Super-Signal West Pico Chemiluminescent substrate (Pierce Chemical Co., Rockford, IL, USA) or Immobilon Western Chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA, USA). Signal intensities were analyzed using an LAS-3000 mini imaging analyzer and Multi Gauge software, version 3.0 (Fujifilm, Tokyo, Japan).
8
Protein Extraction and Western Blot Analysis
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Heads were removed from newly eclosed animals and homogenized in 25 μl solubilization buffer, (60 mM Tris-HCl, pH 6.8, 20%SDS, 0.0004% bromophenol blue, 10% ß-mercaptoethanol, 20% glycerol) and diluted with 25 μl H2O. The homogenate was incubated 1 hour at 37°C and loaded onto a NuPAGE 4–12% Bis-Tris Gel (Novex) and size fractionated. Proteins were electroblotted to Immobilon-P polyvinylidene difluoride membranes (Millipore) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Membranes were blocked (5% BioRad Blotting-Grade blocker in PBS) for 1 hr, and primary anti-RTP antibody 1:2000 in blocking buffer was added and incubated overnight at 4°C. Membranes were washed 3X for 5 minutes in TBST pH 7.4. Secondary antibody, goat anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare) in blocking buffer (1:3000) was added for 1 hour, then washed 3X for 5 minutes pH 7.4. Detection was with ECL HRP chemiluminescence as specified by the manufacturer (Invitrogen Novex). RTP antibody was generated against the peptide GFPRNEGFFQDCRFMR. Peptide preparation, rabbit immunization, and antibody affinity purification was carried out by Biomatik (Cambridge, ON).
9
Western Blot Analysis of EGFP Expression
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Twenty-four hours post-transfection with DNA encoding the indicated plasmid, N2As were lysed in 2× protein sample buffer (150 µL). Cell lysates were subjected to SDS-PAGE, transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA); after a 60 min, incubation in blocking solution (2% in 0.1% TBST) membranes were incubated with rat α-EGFP-monoclonal IgG (3H9, Chromotek, Planegg, DE, 1:5000 final dilution) or mouse α-β-actin monoclonal IgG (A2228, Millipore Sigma, Mass, USA, 1:20,000 final dilution). Bound primary antibodies were recognized as α-rat or α-mouse IgG secondary antibody conjugated with horseradish peroxidase (Life Technologies, MA, USA, 1:1000), and secondary antibodies were visualized using Super Signal™ West Pico PLUS Chemiluminescent Substrate (34578, Thermo, IL, USA) on an Azure C600 Imaging System (Dublin, CA, USA), using a 60 s exposure.
10
Western Blot Analysis of Cell Lysates
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Cells and spheroids lysates were prepared, and an equal concentration of protein was loaded to polyacrylamide gel electrophoresis and blotted onto Immobilon-p polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Antibodies (Table 1 were purchased from Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA), Mitenyl Biotec (Auburn, CA, USA), BD Biosciences (San Jose, CA, USA), and Abcam (Cambridge, MA, USA), and specific proteins were detected by the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA). The list of antibodies is shown in Table 1 .
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