Brain homogenates were stored with 1% IGEPAL-CA, 0.5% Sodium deoxycholate, 0.1% SDS and 1% protease inhibitor cocktail (Sigma, cat no. P-8340). Samples were resolved using 12% SDS-PAGE and transferred to nitrocellulose membrane (biorad) using semidry transfer apparatus. MBP was probed with SMI-94 antibody (Cambridge bioscience) and B-actin antibody (Sigma, a3854) and immunoreactivity was detected using Amersham ECL substrate (GE Healthcare, RPN2232).
Amersham ecl substrate
Manufactured by GE Healthcare
Sourced in United Kingdom
Amersham ECL substrate is a chemiluminescent detection reagent for Western blotting. It produces a luminescent signal when combined with a horseradish peroxidase (HRP)-labeled detection system, allowing the visualization of target proteins.
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Lab products found in correlation
Amersham ecl, by Cytiva (5 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti spry1, by Cell Signaling Technology (1 mentions)
Versadoc imaging system model 4000, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
G box f3, by Syngene (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ms safe protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Mouse anti stat3, by Cell Signaling Technology (1 mentions)
Rabbit anti p stat3 tyr705, by Cell Signaling Technology (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Microchemi 4.2 system, by DNR Bio-Imaging Systems (1 mentions)
5 protocols using amersham ecl substrate
1
Western Blot Analysis of MBP in Brain Homogenates
2
Western Blotting of Sprouty1 Protein
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Protein extracts from tissues were obtained by mechanical lysis using denaturing lysis buffer (50 mM HEPES, 2% SDS), boiled and sonicated. Protein was electrophoresed on 10% SDS polyacrylamide gels, transferred onto PVDF membrane (Millipore) and blocked using 3% BSA (Sigma) in TBS-Tween (0.1%, TBST) for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4 ºC. Signal was detected using Horseradish Peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch), followed by a chemiluminescence reaction using Amersham ECL substrate (GE Healthcare). Chemiluminescent signal was detected using the VersaDoc Imaging system Model 4000 (Bio-Rad), and densitometry was performed using its software package (ImageLab, Bio-Rad). Primary antibodies were Anti-Spry1 (Cell Signalling, Cat# 13,013 or Cat# 12,993) and Anti β-actin (Santa Cruz, sc-1616).
3
Western Blot Protein Analysis Protocol
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Equal amounts of protein (10–20 μg/well) were loaded and separated using SDS-PAGE (7–15%). Proteins were transferred to PVDF membranes and blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween (TBS-T). Blots were incubated overnight with primary antibodies (1:1000) at 4° C, washed in TBS-T and incubated with secondary antibodies (1:10,000) at room temperature for 1 h. Protein bands were visualized with a Amersham ECL substrate (GE Healthcare Bio-Sciences, PA) using a UVP Bioimaging System (Upland, CA). Image capturing and analysis was performed with Vision Works imaging software (Upland, CA). Optical densities obtained for all proteins were normalized to their respective α-tubulin or actin loading controls and the data is expressed as percentage over control levels.
4
Western Blot Analysis of N-Glycan Composition
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Plant leaf tissue was homogenized in 2 μl 1 × PBS per mg LFW using 3 mm chrome steel ball bearings and a Mixer Mill MM400 (Retsch, Castleford, United Kingdom). Crude extract was centrifuged at 20,000×g for 15 min and clarified extracts were resolved on a NuPage 4%–12% Bis-Tris gel (Life Technologies, Paisley, United Kingdom) and transferred onto a nitrocellulose membrane. The membrane was blocked with blocking buffer containing 5% skimmed milk in 1 × TBS + 0.05% Tween.
To analyze the fucosyl- and xylosyl N-glycan composition in total soluble protein, blots were incubated with either 1:5,000 rabbit anti-xylose or anti-fucose antisera (both from Agrisera, Sweden), followed by 1:10,000 peroxidase-conjugated polyclonal anti-rabbit antisera (Sigma, United States). Detection was performed using Amersham ECL substrate (GE Healthcare, United Kingdom) visualized by a G:BOX F3 (Syngene, United Kingdom).
To analyze the fucosyl- and xylosyl N-glycan composition of purified recombinant IgG, 200 ng protein were loaded onto a NuPage 4%–12% Bis-Tris gel. SDS-PAGE and Western blotting was performed as described above. Membranes were subsequently probed 1:5,000 with HRP-labeled anti-IgG H + L chain antibody (31410, Thermo Fisher Scientific, United States) or anti-xylose and anti-fucose antibodies and developed as above.
To analyze the fucosyl- and xylosyl N-glycan composition in total soluble protein, blots were incubated with either 1:5,000 rabbit anti-xylose or anti-fucose antisera (both from Agrisera, Sweden), followed by 1:10,000 peroxidase-conjugated polyclonal anti-rabbit antisera (Sigma, United States). Detection was performed using Amersham ECL substrate (GE Healthcare, United Kingdom) visualized by a G:BOX F3 (Syngene, United Kingdom).
To analyze the fucosyl- and xylosyl N-glycan composition of purified recombinant IgG, 200 ng protein were loaded onto a NuPage 4%–12% Bis-Tris gel. SDS-PAGE and Western blotting was performed as described above. Membranes were subsequently probed 1:5,000 with HRP-labeled anti-IgG H + L chain antibody (31410, Thermo Fisher Scientific, United States) or anti-xylose and anti-fucose antibodies and developed as above.
5
Immunoblotting for Phosphorylated STAT3
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V-SVZ tissue was lysed in radioimmunoprecipitation buffer containing 1× MS-Safe protease and phosphatase inhibitor cocktail (Sigma). Total protein (30 μg) was resolved using 4%–15% gradient Mini-PROTEAN TGX gels (Bio-Rad) according to the manufacturer's protocol, was transferred to nitrocellulose membranes, blocked in 5% BSA (GE Healthcare) and incubated with rabbit anti-p-STAT3 (Tyr705, Cell Signaling Technology, 1:1,000, catalog no. 9145) and mouse anti-STAT3 (Cell Signaling Technology, 1:1,000, catalog no. 9139). Signal was detected using horseradish peroxidase-conjugated secondary antibodies (GE Healthcare), followed by chemiluminescence detection with Amersham ECL substrate (GE Healthcare) and the MicroChemi4.2 system (DNR Bio-Imaging Systems).
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