Embryonic pancreases were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% SDS, 1% NP40 and 0.5% Sodium Deoxycholate) and extraction of histones was optimized through the addition of HCl to a final concentration of 0.2 N. After 30 min incubation on ice, cell debris was pelleted and supernatants were quantified and prepared for SDS-PAGE electrophoresis on 16% Tris-tricine homemade gels. Proteins were then transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA) and incubated overnight at 4 °C with the antibodies indicated Table S3 . Blots were visualized with ECL Reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY). Protein spots were quantitated with Image J software (http://rsb.info.nih.gov/ij/index.html ).
Polyscreen pvdf membrane
Manufactured by PerkinElmer
Sourced in United States
The Polyscreen PVDF membrane is a microporous membrane made of polyvinylidene fluoride (PVDF) material. It is designed for use in various laboratory applications requiring high-performance filtration and separation. The membrane provides efficient retention of particles and macromolecules while allowing the passage of liquid or gas samples.
Automatically generated - may contain errors
Lab products found in correlation
Las4000 lumi imager, by Fujifilm (4 mentions)
Ecl reagent, by Thermo Fisher Scientific (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
Lowry protein assay kit, by Bio-Rad (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Anti s6, by Cell Signaling Technology (1 mentions)
Anti ps6, by Cell Signaling Technology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
6 protocols using polyscreen pvdf membrane
1
Histone Extraction from Embryonic Pancreases
2
Quantitative Western Blotting of Islet Proteins
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Islets were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% (w/v) SDS, 1% (v/v) NP40 and 0.5% (w/v) sodium deoxycholate). After 3 consecutive cycles of dry ice and 37 °C, cell debris was pelleted and supernatants were prepared for SDS-PAGE electrophoresis on 8% Tris-tricine homemade gels. Proteins were then transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA) and incubated overnight at 4 °C with the antibodies indicated in Supplementary Table 2 . Immunoblots were developed with horseradish peroxidase-conjugated secondary antibodies (1:5000, GE Healthcare Bio-Sciences Corp. Piscataway, NJ, USA) and visualized with ECL Reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY). Protein spots were quantitated with Image J software. Due to the little amount of protein obtained from islets, protein extracts were not quantified but instead, the same number of islets was loaded in each lane. Representative images of the quantification results were selected. Uncropped blots are supplied in the Source Data file.
3
Western Blot Analysis of Protein Samples
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VAT samples from twelve subjects from each group were lysed using RIPA lysis and extraction buffer and centrifuged at 18,000× g, 4 °C for 20 min. A total of 20 µg of protein was resolved by SDS-PAGE (10%) and then transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA), blocked in 5% non-fat milk powder added to TRIS-buffered saline with 0.05% Tween 20 (TBST) and further incubated overnight at 4 °C with the primary antibodies cited above at 1:1000 dilution. Actin (A2066, Sigma-Aldrich; 1:1000) was used as loading control. Following a wash in TBST solution, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (NA934, GE Healthcare, Chicago, IL, USA) or anti-mouse (NA931, GE Healthcare) antibodies and visualized with Immobilon Forte western HRP substrate (Millipore, Burlington, MA, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY, USA). Protein spots were quantitated with Image J software.
4
Western Blot of Islet Proteins
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Freshly isolated or cultured islets were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% SDS, 1% IGEPAL-CA630, 0.5% Sodium Deoxycholate, protease and phosphatase inhibitors), followed by sonication and frozen-thaw cycles. Protein concentration was measured with the Lowry protein assay kit (Bio-Rad, Hercules, CA, USA). Protein extracts (15–30 μg per replicate) were separated by SDS-PAGE electrophoresis onto 7.5–10% Tris-tricine gels and transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA). Membranes were blocked for 1 h with TBS-0.05%Tween-5% BSA solution, followed by overnight incubation at 4 °C with the corresponding primary antibodies diluted in TBS.0.05%Tween: rabbit anti-MafA (1:250, Novus Biologicals), rabbit anti-p-Creb (S133), anti-Creb, anti-p-Erk1/2 (T202,Y204), anti-Erk, anti-p-Akt (T308), anti-Akt, anti-p-S6 (S235,S236), anti-S6 (1:1000, Cell Signaling Technology), and mouse anti-α-tubulin (1:1000; Sigma–Aldrich) as a loading control. Blots were visualized with ECL Reagent (Pierce Biotechnology, Rockford, USA) using a LAS4000 Lumi-Imager (Fuji Photo Fil, Valhalla, NY). Protein spots were quantified with Image Studio Lite V5.2 software.
5
Quantitative Western Blot Analysis of Viral Proteins
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Total soluble proteins from 0.05 g infiltrated or inoculated leaves were extracted by grinding the tissues in 3X protein sample buffer (3% SDS, 30% glycerol, 0.01% bromophenol blue, and 15% β-mercaptoethanol.). The samples were incubated at 100°C for 5 min and then the supernatant was collected after centrifugation (8000xg, 3 min). The proteins were resolved on a 12% sodium dodecyl sulphate-polyacrylamide gel and transferred to Polyscreen PVDF membrane (PerkinElmer, Waltham, MA, USA). Membranes were probed using corresponding antibodies, i.e., monoclonal (MAb) and polyclonal antibodies (PAb) to WSMoV NSs [45 (link)]; PAb to ZYMV HC-Pro [47 (link)], ZYMV coat protein (CP) [50 ] or GFP [51 (link)]. Membrane was stained with Coomassie Brilliant Blue R250 and amount of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was used as a loading control. Banding signal intensity was quantified by Kodak Image system 4000MM (EASTMAN Kodak Company, Rochester, NY, USA).
6
Western Blot Analysis of Pancreatic Lysates
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Cells and embryonic pancreases were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% SDS, 1% NP40 and 0.5% Sodium Deoxycholate). 50 μg of lysates were separated by PAGE-SDS electrophoresis, transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA) and incubated overnight at 4 °C with the antibodies indicated in Supplementary Table S3 . Blots were visualized with ECL Reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY). Protein spots were quantitated with Image J software (http://rsb.info.nih.gov/ij/index.html ).
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