Total protein of 200 mg of grinded mycelia was extracted as described by Apostolaki et al. [56 (link)] and protein concentration was determined by the Bradford assay. Total proteins (50 µg) were separated by SDS-PAGE (10% (w/v) polyacrylamide gel) and electroblotted (OmniPAGE Electroblotting Units, Cleaver Scientific Ltd) onto a nitrocellulose membrane (0.45 µm, Thermo Scientific). Membrane was then incubated in stripping buffer (10 mM Tris–HCl pH 6.8, 2% SDS and 100 mM β-mercaptoethanol) for 15 min at 55°C (as described by Kaur & Bachhawat [57 (link)]). Membrane was then treated with 5% non-fat dry milk, and immunodetection was done with a primary mouse anti-GFP monoclonal antibody (Roche Applied Science), or a mouse anti-actin monoclonal (C4) antibody (MP Biomedicals) and a secondary sheep anti-mouse IgG HRP-linked antibody (GE Healthcare). Blots were developed using the Amersham ECL Western blotting detection reagents and analysis system (GE Healthcare), and images were acquired using the Gene Sys software from the GBoxChemi XT4 System (Syngene).
Amersham ecl western blotting detection reagents and analysis system
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The Amersham ECL Western blotting detection reagents and analysis system is a lab equipment product from GE Healthcare that is used for the detection and analysis of proteins in Western blotting experiments. The system includes reagents and tools for chemiluminescent detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Amersham ecl western blotting, by Cytiva (6 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Cell Signaling Technology (2 mentions)
Bcl 2, by Abcam (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Gbox chemi xt4 system, by Syngene (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Mouse anti actin monoclonal c4 antibody, by MP Biomedicals (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 camera, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Aprotinin, by Nacalai Tesque (1 mentions)
β actin, by Merck Group (1 mentions)
Leupeptin, by Nacalai Tesque (1 mentions)
Sheep anti mouse igg hrp conjugate, by GE Healthcare (1 mentions)
Protease and phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
6 protocols using amersham ecl western blotting detection reagents and analysis system
1
Immunoblotting of Fungal Proteins
2
Western Blot Analysis of tPA and MPs
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Recombinant tPA and MPs samples (tPA-transfected and control, ctrl-MPs coming from HEK supernatants) were resolved for the western blot on 4-12 % Nupage gels (Invitrogen) gels in reduced conditions and transferred onto iBlot, a polyvinylidene difluoride (PVDF) membrane by iBlot transfer stacks (Invitrogen). Blots were blocked with 5% BSA (bovine serum albumin, Sigma-Aldrich, L'Isle d'Abeau, France) in Tris-buffered saline containing 0.05% Tween-20 and then incubated overnight with the same buffer at 1% BSA with anti-tPA antibody (1/5000), followed by incubation with a peroxidase-conjugated goat anti-sheep secondary antibody (1/50000, sigma) and developed by a Amersham ECL Western blotting detection reagents and analysis system (GE Healthcare, France) using ImageQuant™ LAS 4000 camera (GE healthcare, France).
3
Rat brain protein extraction and immunoblotting
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Brain samples were obtained from Sv2aL174Q or F344 rats under pentobarbital (80 mg/kg, i.p.) anesthesia and chilled in ice-cold saline. Brains were then dissected into various region blocks and homogenized in an ice-cold lysis buffer (pH 7.5) containing: (in mM) Tris 20, NaCl 150, MgCl2 10, EDTA 1.0, 1% Triton X-100 and a mixture of protease inhibitors (leupeptin, aprotinin, E-64, pepstatin A, bestatin and 4-(2-Aminoethyl) benzensulfonyl fluoride hydrochloride) (Nakarai Tesque, Tokyo, Japan). The homogenate was centrifuged at 15,000 g, 4 °C for 30 min and the supernatant was stored at −80 °C for subsequent analysis.
Western blotting was performed according to the methods described previously14 (link)23 (link)24 (link), using the corresponding primary antibodies as follows, mouse monoclonal antibody against Stxbp1 (1:2000, Synaptic Systems), Nsf (1:2000, Synaptic Systems), Napa (1:2000, Santa Cruz Biotechnology), Syt1 (1:5000, Synaptic Systems) and β-actin (dilution 1:2000, Sigma Aldrich, St Louis, MO) and a sheep anti-mouse IgG-HRP conjugate (dilution 1:2000, GE Healthcare) as the secondary antibodies. Final detection was performed with the enhanced chemiluminescence method (Amersham ECL Western blotting detection reagents and analysis system, GE healthcare, Buckinghamshire, UK) using a lumino imaging analyzer (LAS-3000, FUJIFILM, Tokyo, Japan).
Western blotting was performed according to the methods described previously14 (link)23 (link)24 (link), using the corresponding primary antibodies as follows, mouse monoclonal antibody against Stxbp1 (1:2000, Synaptic Systems), Nsf (1:2000, Synaptic Systems), Napa (1:2000, Santa Cruz Biotechnology), Syt1 (1:5000, Synaptic Systems) and β-actin (dilution 1:2000, Sigma Aldrich, St Louis, MO) and a sheep anti-mouse IgG-HRP conjugate (dilution 1:2000, GE Healthcare) as the secondary antibodies. Final detection was performed with the enhanced chemiluminescence method (Amersham ECL Western blotting detection reagents and analysis system, GE healthcare, Buckinghamshire, UK) using a lumino imaging analyzer (LAS-3000, FUJIFILM, Tokyo, Japan).
4
Investigating Thermal Stress on Glioblastoma
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Glioblastoma cell lines (U87MG and T98G) were cultured under standard conditions, as previously mentioned. These cell lines were similarly incubated at 34°C and 40°C for 72h. Cells were lysed using a sucrose lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Cell Signaling). Protein concentrations were measured based at the BCA protein assay kit (Thermo Scientific Pierce, USA). Specific rabbit polyclonal primary antibodies were used for western blot analysis, anti-HSP90 (1:1000; ab13495, Abcam) and anti-Caspase9 (1:1000; ab47537, Abcam).
Whole fraction samples were separated on discontinuous SDS gels using 10% separating and 5% stacking gels. Forty micrograms of the cell extracts were loaded on the gel. Immunoblotting was performed utilizing PVDF-PSQ membranes (Millipore Corp.). Following a blocking step with 5% non-fat dry milk in 150 mM NaCl, 10 mM Tris, pH 7.5 containing 0.1% (v/v) Tween 20 (TBS-T) at room temperature (RT) for 2h, the membranes were hybridized overnight at 4°C with the primary antibodies. The membranes were then hybridized for 2h at 37°C with the secondary antibody, goat polyclonal to rabbit IgG (H+L)-HRP (1:3.000, Biorad, 1706515, USA) and finally developed in Amersham ECL Western blotting detection reagents and analysis system (RPN2209, GE Healthcare) utilizing Chemidoc MP Imaging System (Biorad, USA).
Whole fraction samples were separated on discontinuous SDS gels using 10% separating and 5% stacking gels. Forty micrograms of the cell extracts were loaded on the gel. Immunoblotting was performed utilizing PVDF-PSQ membranes (Millipore Corp.). Following a blocking step with 5% non-fat dry milk in 150 mM NaCl, 10 mM Tris, pH 7.5 containing 0.1% (v/v) Tween 20 (TBS-T) at room temperature (RT) for 2h, the membranes were hybridized overnight at 4°C with the primary antibodies. The membranes were then hybridized for 2h at 37°C with the secondary antibody, goat polyclonal to rabbit IgG (H+L)-HRP (1:3.000, Biorad, 1706515, USA) and finally developed in Amersham ECL Western blotting detection reagents and analysis system (RPN2209, GE Healthcare) utilizing Chemidoc MP Imaging System (Biorad, USA).
5
Cinobufagin and Cisplatin Combination Therapy
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Cinobufagin and CDDP were purchased from from Sigma (St. Louis, MO, USA). All media were obtained from GE Healthcare (Hyclone, UT, USA). Fetal bovine serum was from Gibco (Australia). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Primary antibody for Notch-1 intracellular domain (NICD1), Hes1, VEGF, Bcl-2 and Bax were from Abcam (Cambridge, UK). RIPA Lysis Buffer, primary antibodies against, Cleaved Caspase-9, Cleaved Caspase-3, MMP-2, MMP-9 and horseradish peroxidase (HRP)-labeled secondary antibody were from Cell Signaling Technology (Beverly, MA, USA). Amersham ECL Western blotting detection reagents and analysis system was purchased from GE Healthcare (Hyclone, UT, USA) and all other chemicals were from Sigma (St. Louis, MO, USA), unless otherwise indicated.
6
Baicalein's Anticancer Mechanisms
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Baicalein, Hoechst 33258, dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Baicalein was dissolved in 100 % DMSO to prepare a 200 mM stock solution, which was stored at −20 °C. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). Primary antibodies against human Bax, Bcl-2, p21, p27, cyclinD1, CDK2, pRb, MMP-2, MMP-9, β-catenin, c-myc, survivin were purchased from Abcam (Cambridge, UK). RIPA Lysis Buffer, primary antibodies against cleaved caspase-9, cleaved caspase-3, cleaved PARP, cytochrome c, GAPDH and horseradish peroxidase (HRP)-labeled secondary antibody were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). Amersham ECL Western blotting detection reagents and analysis system were purchased from GE Healthcare (Buckinghampshire, UK).
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