Membrane blocking agent

Manufactured by GE Healthcare

Sourced in United Kingdom

Membrane Blocking Agent is a laboratory product used to block non-specific protein binding on membranes, such as those used in Western blotting and other immunoassays. It helps reduce background signal and improve the specificity of target protein detection.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (2 mentions) Anti α tubulin antibody, by Santa Cruz Biotechnology (2 mentions) Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions) Anti β actin antibody, by Cell Signaling Technology (2 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Iblot system, by Thermo Fisher Scientific (1 mentions) Nupage antioxidant, by Thermo Fisher Scientific (1 mentions) Chemidox xrs imaging system, by Bio-Rad (1 mentions) Rabbit anti ask1, by Cell Signaling Technology (1 mentions) Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Protein assay, by Bio-Rad (1 mentions) Chemidoc xrs imaging system, by Bio-Rad (1 mentions) Ecl agent, by GE Healthcare (1 mentions) Spectramax m2, by Molecular Devices (1 mentions) Roti block, by Carl Roth (1 mentions) Enhanced chemiluminescence, by GE Healthcare (1 mentions) Af2557, by R&D Systems (1 mentions) Haf016, by R&D Systems (1 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions)

Spelling variants of this product

Blocking agent (10 citations)

8 protocols using membrane blocking agent

Western Blot Protein Analysis Protocol

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Cells were lysed in 1% protease inhibitor cocktail (Roche) containing radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% sodium deoxycholate, and 0.1% SDS), by thorough sonication. Protein lysate (20–40 μg) was mixed with an equal volume of EzApply (ATTO) and boiled at 95 °C for 5 min. Samples were then separated on a 3–8% NuPAGE Novex tris-acetate mini gel (Invitrogen) at 150 V for 75 min with the addition of NuPAGE antioxidant (Invitrogen) during electrophoresis. The fractionated proteins were transferred to a nitrocellulose membrane using an iBlot system (Invitrogen) with the program, P0, 13 min. The membrane was blocked with Membrane Blocking Agent (GE Healthcare life sciences) and incubated with primary antibody at 4 °C overnight. After 3 washes with PBST, the membrane was incubated with secondary antibody for 1 hour at room temperature. Refer Supplementary Table S1 for detailed antibody information.
Detection was carried out with Pierce ECL western blotting substrate (Thermo scientific). Visualisation and semi-quantification of images were performed by a ChemiDox XRS + imaging system (BIO-RAD).

Shoji E., Sakurai H., Nishino T., Nakahata T., Heike T., Awaya T., Fujii N., Manabe Y., Matsuo M, & Sehara-Fujisawa A. (2015). Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells. Scientific Reports, 5, 12831.

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Western Blot Analysis of Protein Lysates

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Ear tissue was lysed in Mammalian Protein Extraction Reagent (MPER, Thermo Fisher, Waltham, MA), and protein concentrations were measured using Bio-Rad Protein Assay (Bio-Rad Laboratories Inc.). Approximately 20 μg of protein were loaded onto an sodium dodecyl sulfate (SDS) polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in Tris Buffered Saline (TBS) with 0.05% Tween 20 (Thermo Fisher, Waltham, MA) and 5% Membrane Blocking Agent (GE Healthcare, Buckinghamshire, UK) for 1 hour while shaking. Overnight incubation at 4°C was performed with antibodies for either mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX) or rabbit anti-ASK1 (Cell Signaling, Danvers, MA) in TBS with 0.05% Tween 20. The primary antibody was removed and the blots were washed three times in TBS with 0.05% Tween 20. Then their respective horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz) were applied to the blots for 1 h at room temperature, washed intensively in TBS with 0.05% Tween 20 and then reacted with ECL Advanced Detection system (Amersham, Pittsburgh, PA) for 5 min at 25°C. Detection of the membranes was done with a FujiFilm Intelligent Darkbox (FujiFilm Co., Tokyo, JP).

Zhang Q.S., Kurpad D.S., Mahoney M.G., Steinbeck M.J, & Freeman T.A. (2017). Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration. PLoS ONE, 12(10), e0185803.

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Acute Sacrificing and Western Blot Analysis of S6 Ribosomal Protein in Mouse ACC

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Mice (male; 2 to 3 months of age; n = 6 per group) were acutely sacrificed by decapitation. The brain (the ACC) was then dissected quickly within 30 s, snap-frozen in liquid nitrogen, and subsequently lysed in radioimmunoprecipitation assay buffer containing 1% Triton X-100, 0.1% SDS, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Sigma-Aldrich). The extracted proteins were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (0.45-μm pore, Millipore), which was incubated in a membrane-blocking agent (GE Healthcare) for 30 min and probed with anti-S6 ribosomal protein (rabbit, 1:1000; Cell Signaling Technology) or anti–phospho-S6 (Ser^235/236) antibodies (rabbit, 1:1000; Cell Signaling Technology) and subsequently with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (1:10,000; GE Healthcare). Signals were detected with an ECL agent (GE Healthcare). Blotting images were acquired by ChemiDoc XRS imaging system (Bio-Rad), and the densitometric analysis was performed using ImageJ (NIH).

Sumitomo A., Horike K., Hirai K., Butcher N., Boot E., Sakurai T., Nucifora FC J.r., Bassett A.S., Sawa A, & Tomoda T. (2018). A mouse model of 22q11.2 deletions: Molecular and behavioral signatures of Parkinson’s disease and schizophrenia. Science Advances, 4(8), eaar6637.

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Quantifying Secretagogin in Plasma and Samples

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A human secretagogin ELISA (BioVendor, Brno, Czech Republic) was used. The plates were blocked with a solution of membrane blocking agent (1%, GE Healthcare) and Rotiblock (1× dilution, Carl Roth GmbH), rinsed with provided kit wash and the manufacturer’s instructions were followed. The plasma samples were diluted three-fold, media samples five-fold, and EndoC extracts 100-fold, prior to analysis. Insulin, human C-peptide and proinsulin ELISAs were used for measurements in plasma, according to the manufacturer’s instructions (Mercodia, Uppsala, Sweden). All samples were assayed in duplicates, randomized and blinded during the run. All ELISA results were obtained using a microplate reader (SpectraMax M2, Molecular devices, Ca, USA).

Hansson S.F., Zhou A.X., Vachet P., Eriksson J.W., Pereira M.J., Skrtic S., Jongsma Wallin H., Ericsson-Dahlstrand A., Karlsson D., Ahnmark A., Sörhede Winzell M., Magnone M.C, & Davidsson P. (2018). Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction. PLoS ONE, 13(4), e0196601.

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Western Blot Analysis of PGRN

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For PGRN protein analysis, supernatants were mixed with equal amount of in 2× sample buffer boiled for 5 min, and resolved on a 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane and blocked over-night with membrane blocking agent (GE Healthcare) at 4°C. The blots were incubated in PBST with 1:250 anti-mouse PGRN polyclonal antibody (R&D Systems AF2557) for 1hour followed by extensive washing. After incubating with horseradish peroxidase-conjugated anti-sheep IgG secondary antibody (R&D Systems HAF016; 1:4,000) at room temperature for 1 hour, blots were visualized using enhanced chemiluminescence (GE Healthcare) and a Bio-Rad ChemiDoc MP Image System (Bio-Rad Laboratories, ON Canada). The same blot was stained with mouse monoclonal β-actin antibody (Sigma AC-40; 1:1000), as a control for total protein loading.

Van Kampen J.M, & Kay D.G. (2017). Progranulin gene delivery reduces plaque burden and synaptic atrophy in a mouse model of Alzheimer's disease. PLoS ONE, 12(8), e0182896.

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Western Blot Analysis of Phosphorylated eNOS

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A total of 40 μg of proteins were separated by 4%–12% pre-cast polyacrylamide gel (NuPAGE^® Novex^® 4%–12% Bis-Tris Gels, Thermo Scientific) and transferred to nitrocellulose filters. Membranes were blocked with 5% skim milk (Membrane Blocking Agent, GE Healthcare, Chalfont St Giles, UK) in Tris-buffered saline solution containing 0.1% Tween-20 and incubated overnight at 4 °C with rabbit anti-human phospho-eNOS^Ser1177 (1:1000), eNOS (1:1000) or β-actin (1:10,000) (Cell Signaling Technology, Boston, MA, USA) antibodies. Membranes were then incubated with Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Cell Signaling) and detected by chemiluminescence using ECL western Blotting Detection Reagent (GE Healthcare, Chalfont St Giles, UK). Images were scanned and quantified using ImageJ software (Version 1.48, National Institutes of Health, MD, USA). Activation of eNOS was expressed as a ratio of p-eNOS to eNOS (which detects both phosphorylated and non-phosphorylated forms).

Spigoni V., Mena P., Cito M., Fantuzzi F., Bonadonna R.C., Brighenti F., Dei Cas A, & Del Rio D. (2016). Effects on Nitric Oxide Production of Urolithins, Gut-Derived Ellagitannin Metabolites, in Human Aortic Endothelial Cells. Molecules, 21(8), 1009.

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Analyzing TGF-β-induced Nuclear Signaling

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Cells were lysed in Laemmli Sample buffer (Bio‐Rad, Hercules, CA, USA) containing DTT and 1% protease inhibitor cocktail. Nuclear extracts at 24 h after TGF‐β (R&D Systems, Minneapolis, MN, USA) treatment or at 48 h after TGF‐β receptor inhibitor (A‐83‐01, Wako, Tokyo, Japan) treatment were prepared by using NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). The samples were separated using 12% SDS‐PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% membrane blocking agent (GE Healthcare, Buckinghamshire, UK) in PBS, and probed with anti‐SIX1 antibody (1:250, Acris Antibodies GmbH, Herford, Germany) or anti‐phospho‐smad2/3 antibody (1:500, Cell Signaling Technology, Danver, MA, USA) at 4°C overnight, anti‐α tubulin antibody (1:1000; Santa Cruz, Dallas, Tx, USA) at room temperature for 1 h or anti‐β actin antibody (1:2000; Cell Signaling Technology) at room temperature for 2 h, then washed and incubated with HRP‐conjugated anti‐rabbit immunoglobulin (DAKO) at room temperature for 2 h. Immunoreactive protein bands were identified with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).

Nishimura T., Tamaoki M., Komatsuzaki R., Oue N., Taniguchi H., Komatsu M., Aoyagi K., Minashi K., Chiwaki F., Shinohara H., Tachimori Y., Yasui W., Muto M., Yoshida T., Sakai Y, & Sasaki H. (2017). SIX1 maintains tumor basal cells via transforming growth factor‐β pathway and associates with poor prognosis in esophageal cancer. Cancer Science, 108(2), 216-225.

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Western Blot Analysis of Protein Expression

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Samples were separated using SDS‐PAGE (7.5% acrylamide). Proteins were transferred to a nitrocellulose membrane and blocked with 5% membrane blocking agent (GE Healthcare, Buckinghamshire, UK) in PBS, and probed with anti‐SIM2 antibody (1:250; Abcam, Cambridge, UK), anti‐ARNT antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐SOD2 antibody (1:5000; Abcam), or anti‐β‐actin antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight, or anti‐α tubulin antibody (1:1000; Santa Cruz Biotechnology) at room temperature for 2 hours, then washed and incubated with HRP‐conjugated antigoat immunoglobulin (Dako, Carpinteria, CA, USA) or HRP‐conjugated antimouse immunoglobulin (Dako) at room temperature for 2 hours. Immunoreactive protein bands were identified with Pierce ECL Plus Western Blotting Substrate (Thermo Scientific).

Tamaoki M., Komatsuzaki R., Komatsu M., Minashi K., Aoyagi K., Nishimura T., Chiwaki F., Hiroki T., Daiko H., Morishita K., Sakai Y., Seno H., Chiba T., Muto M., Yoshida T, & Sasaki H. (2018). Multiple roles of single‐minded 2 in esophageal squamous cell carcinoma and its clinical implications. Cancer Science, 109(4), 1121-1134.

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