Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were loaded on a 10% or 12% SDS-gel, resolved using SDS-PAGE and transferred to a nitrocellulose membrane (AmershamTM ProtranTM; Cytiva, Korea). Membranes containing transferred proteins were incubated with antibodies against cyclin-D1 (1:1,000, Cat. No. 2978; Cell Signaling Technology), cyclin-D3 (1:1,000, Cat. No. 2936; Cell Signaling Technology), p18 (1:1,000, Cat. No. 2896; Cell Signaling Technology), p21 (1:1,000, Cat. No. 2947; Cell Signaling Technology), and β-actin (1:5,000, Cat. No. A2066; Sigma-Aldrich) overnight at 4°C. The following day, membranes were washed with PBS-Tween and incubated for 1 h at room temperature with the appropriate peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA). Membranes were then washed with PBS-Tween and signals were visualized using EZ-Western Lumi Pico solution (DoGenBio). Digital imaging was performed using imageQuant LAS4000 (Cytiva).
Amersham protran
Manufactured by Cytiva
Sourced in United States, Germany
Amersham Protran is a nitrocellulose membrane designed for protein transfer and detection in Western blotting applications. It provides consistent and reliable performance for the effective transfer and immobilization of proteins from polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Cyclin d3, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Imagequant las 4000, by Cytiva (1 mentions)
Image lab software version 6, by Bio-Rad (1 mentions)
Hybri slot apparatus, by Thermo Fisher Scientific (1 mentions)
Anti snap25, by Cell Signaling Technology (1 mentions)
Anti epcam, by Abcam (1 mentions)
Cell lytic m, by Merck Group (1 mentions)
Amersham ecl western blotting detection reagent, by Cytiva (1 mentions)
Anti actin, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated antibody, by Bio-Rad (1 mentions)
Nzy supreme ecl hrp substrate, by NZYTech (1 mentions)
Mouse monoclonal anti polyhistidine antibody, by Merck Group (1 mentions)
Mouse monoclonal anti mbp antibody, by New England Biolabs (1 mentions)
Power blotter apparatus, by Thermo Fisher Scientific (1 mentions)
10 protocols using amersham protran
1
Quantitative Western Blot Analysis
2
Quantification of T-antigen Expression
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Stool proteins (50 μg) were transferred in duplicates onto a nitrocellulose membrane pore size 0.45 μm (AmershamTMProtranTM, Cytiva, Marlborough, MA, USA), using a Hybri-slot apparatus (21052-014; Gibco BRL, Life Technologies, Waltham, MA, USA) under a stabilized vacuum of −0.2 bar. Proteins extracted from T-antigen expressingT24 GCNT1 KO cells were used as positive controls, whereas BSA was used as a negative control. Protein loads were determined by Ponceau S. Nonspecific bindings were blocked with a Carbo-Free Blocking Solution for 1.5 h at RT. Subsequently, the T-antigen expression was evaluated using biotinylated PNA (VerctorLabs, Newark, CA, USA) at a dilution of 1:10,000 in a mixture of 50% CarboFree and 50% PBS 1x for 1 h at RT. Membranes were washed with PBS-T (PSB with 1% Tween 20) and incubated with the VECTASTAIN® Elite ABD-HRP Reagent (1:10; Vector Laboratories, Newark, CA, USA) for 15 min at RT. The Amersham ECL Prime Detection Reagent was used as a developing reagent. Data analysis was performed through Image Lab Software (version 6.0.1) (Bio-Rad, Hercules, CA, USA) in a ChemiDoc XRS (Bio-Rad, CA, USA).
3
His6-PA3973 Cross-linking Assay
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The purified His6-PA3973 was cross-linked with increasing concentrations of glutaraldehyde as previously described [67 (link)]. Samples were then suspended in the loading buffer [50 mM Tris-HCl (pH 8.0), 0.1 M DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol], boiled, and separated on an SDS-PAGE gel, transferred to nitrocellulose membrane (Amersham Protran, Cytiva, Germany), and used in a Western blot analysis with the use of anti-His6 antibodies.
4
Western Blot Analysis of Tumor Cell Proteins
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For western blotting, tumour cells were washed and lysed with CellLytic M (Sigma-Aldrich) containing protease and phosphatase inhibitors. The lysate was cleared by centrifugation at 15,000 rpm for 15 min and denatured at 95 °C for 5 min. The lysate was stored at −80 °C until immunoblot analysis. Proteins were separated by electrophoresis using a NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific) and transferred to Amersham Protran (Cytiva). After blocking the membrane with 5% skim milk for 1 h, it was incubated overnight at 4 °C with specific primary antibodies. After hybridisation with secondary antibodies, the protein bands were visualised using Amersham ECL western blotting detection reagent (Cytiva). The antibodies used in this study are listed as follows; anti-EPCAM (#ab223582, Abcam), anti-SNAP25 (#5308, Cell Signaling Technology), anti-Actin (#MA5-11869, Invitrogen), anti-rabbit IgG (#7074, Cell Signaling Technology), and anti-mouse IgG (#7076, Cell Signaling Technology).
5
Western Blot Protein Extraction and Analysis
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Total protein was extracted in RIPA buffer (1.8 mM NaH2PO4, 8.4 mM Na2HPO4, 0.1% (w/v) SDS, 1.0% (v/v) TritonX 100, 0.1 M NaCl, 0.5% sodium deoxycholate, 1 mM PMSF) supplemented with protease and phosphatase inhibitors. Equal amounts of protein measured by a BCA protein assay kit were separated by SDS-PAGE gel electrophoresis and transferred onto nitrocellulose membranes (Amersham Protran®, Cytiva, Freiburg im Breisgau, Germany). Blots were developed by enhanced chemiluminescence reaction (ECLTM Western blotting Detection reagents, Cytiva). Equal loading was confirmed by reprobing blots with β-actin, GAPDH, or hsc70 antibodies. To avoid membrane striping whenever possible, blots were cut and each strip was incubated with different antibodies to detect several proteins in the same membrane. Proteins from at least three different animals per experimental group were isolated and analyzed by Western blot. For protein quantification, Image J software 1.54 was used; each blot was normalized against β-actin, GAPDH, or hsc70, and plotted as fold change compared to controls.
6
Western Blot Analysis of CstC and β-Tubulin
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Total proteins were harvested using Laemmli buffer (Sigma-Aldrich) and heated at 95°C for 5 min. Samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 15% polyacrylamide gel and transferred to 0.2-μm pore nitrocellulose membrane (Amersham Protran, Cytiva). Membrane was blocked in 5% low-fat milk PBS with 0.1% Tween 20. Following blocking, the membrane was incubated in 1:2,000 dilution of primary antibodies specific for CstC (Cat # ABC20, Sigma-Aldrich) and β-tubulin (Cat # ab6046, Abcam) overnight. Membranes were washed in PBS-Tween and incubated with secondary horseradish peroxidase (HRP)-conjugated antibody (Cat # 1706515, Bio-Rad) for 1 h. Bands were visualized by chemiluminescence using NZY Supreme ECL HRP substrate (Cat # MB19301, NZYTech) in a ChemiDoc XRS (Bio-Rad). Quantification of band intensity was performed on ImageJ software.
7
Cross-linking Analysis of PA2577-His6
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Purified PA2577-His6 was cross-linked with increasing concentrations of glutaraldehyde as previously described [83 (link)]. Samples were then suspended in the loading buffer (50 mM Tris-HCl (pH = 8.0), 0.1 M DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), boiled for 5 min and separated on 12% (w/v) SDS-PAGE gels. After overnight wet transfer onto nitrocellulose membranes (Amersham Protran, Cytiva, Marlborough, MA, USA), Western blot analysis was performed with anti-His6 mouse antibodies.
8
Western Blotting of Polyhistidine-Tagged Proteins
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Proteins were loaded on 12% polyacrylamide gel, with color Prestained Protein Standard (NEB). After migration, proteins were stained with Instant Blue (Abcam). For Western blotting, proteins were transferred onto a nitrocellulose membrane (Amersham Protran, Cytiva) using a Power Blotter apparatus (Invitrogen). Membranes were then saturated in PBS containing 0.1% Tween 20 (PBST) and 4 % skim milk overnight. They were incubated for 2 h with PBST containing a 1:2000 dilution of mouse monoclonal anti-polyHistidine antibody (Sigma-Aldrich) or alternatively with mouse monoclonal anti-MBP antibody (NEB), and then with PBST containing a 1:1000 dilution of Goat anti-mouse IgG Peroxydase conjugated (Invitrogen) for 1 h. Immunodetected proteins were revealed with Pierce ECL Western Blotting Substrate (Thermo), following the manufacturer’s instructions, and chemiluminescence was detected with a ChemiDoc XRS+ system (Bio-Rad).
9
Western Blot Analysis of Cellular Proteins
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All protein lysates were prepared in SDS loading buffer [50 mM Tris HCl pH 6.8, 2% (w/v) SDS and 10% glycerol (v/v), 1% β-mercaptoethanol, 0.02% bromophenol blue] and heated to 70 °C for 5 min before loading. Whole cell (20 μg protein), cytoplasmic (20 μg protein) or nuclear (10 μg protein) lysates were separated by SDS-PAGE on 4% stacking-12% resolving polyacrylamide gels (Mini-PROTEAN Precast Gel system, BioRad, UK). Proteins were transferred to 0.45 μm nitrocellulose (Amersham™ Protran®, Cytiva, Germany) and membranes were blocked with 5% fatty acid-free bovine serum albumin prior to overnight incubation with primary antibodies in blocking buffer (details in Table 1 ) at 4 °C as previously described [38 (link)]. NRF2 and pCRMP2 were detected by the ECL method (detailed in Table 2 ) and all other primary antibodies were detected and quantified using Alexa Fluor™ 680 or IRDye® 800CW-tagged secondary antibodies on a Licor Odyssey CLx (Analysed by Image Studio Lite Version 5.2).
10
Detection of Fasciola gigantica Antigen
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Mouse polyclonal antibodies against rFgNR1+Trx and rTrx were used for the determination of the native protein, FgNR1, in the parasite extracts. An amount of 100 ng of each rFgNR1+Trx or rTrx, 30 μg of each s-CWA and i-CWA, and 10 μg ES of F. gigantica were size separated on 12.5% SDS-PAGE. The proteins were then transferred onto the nitrocellulose membrane (Amersham Protran, Cytiva, Piscataway, NJ, USA) by semidry transferring system (Invitrogen™ Power Blotter, Thermo Fisher Scientific, Carlsbad, CA, USA). Non-specific bindings were blocked using 5% skim milk (Sigma Aldrich, Darmstadt, Germany) for 1 h at room temperature, mouse serum was added in the dilution of 1:400 in antibody diluent (1% BSA in TBS) and incubated at 37°C for 1 h. The membrane was washed several times and then the goat anti-mouse IgG (H+L) secondary antibody, AP conjugate (Invitrogen, Carlsbad, CA, USA), was added and incubated at room temperature for 1 h. Finally, BCIP/NBT substrate was added to the membrane and incubated for a while until the signal could be observed.
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