Western blue stabilized substrate for alkaline phosphatase

Manufactured by Promega

Sourced in United States

Western Blue Stabilized Substrate for Alkaline Phosphatase is a chemiluminescent substrate designed for the detection of alkaline phosphatase-conjugated secondary antibodies in Western blotting applications. The substrate provides a stable, blue-colored signal that can be detected using standard imaging equipment.

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Lab products found in correlation

Control nontargeting sirna 1, by Horizon Discovery (3 mentions) Westernbright sirius, by Advansta (2 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions) Chemidoc mp imaging system, by Bio-Rad (2 mentions) T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Merck Group (2 mentions) A2066, by Merck Group (2 mentions) Sc 858, by Santa Cruz Biotechnology (2 mentions) Sc 368, by Santa Cruz Biotechnology (2 mentions) Amersham imager 600, by GE Healthcare (2 mentions) Ab108319, by Proteintech (2 mentions) Amersham imager, by Cytiva (2 mentions) Chemiimager 4000, by Bio-Techne (2 mentions) Bcl6 sigenome smartpool reagent, by Horizon Discovery (2 mentions) Odyssey fc imager, by LI COR (2 mentions) Anti mouse igg h l alkaline phosphatase conjugated secondary antibodies, by Promega (2 mentions) Anti flag, by Merck Group (1 mentions) Anti gapdh, by Abcam (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Goat anti mouse igg hrp, by Merck Group (1 mentions)

Spelling variants of this product

Alkaline phosphatase (42 citations) Western Blue (17 citations) Western Blue stabilized substrate (13 citations) Western Blue Stabilized alkaline phosphatase substrate (4 citations) Western Blue substrate (4 citations) Stabilized substrate for alkaline phosphatase (2 citations) Alkaline phosphatase substrate (2 citations) Substrates (2 citations)

35 protocols using western blue stabilized substrate for alkaline phosphatase

Western Blot Protein Analysis

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Cell lysates from cell lines were prepared using TNE buffer in the presence of protease inhibitor cocktails (Roche Pharma, Basel, Switzerland). Lysates (100 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 15%) and blotted with the following antibodies: anti-Flag (Sigma-Aldrich, St. Louis, MO, USA), anti-DsRed (Arigo Biolaboratories Corp., Hsinchu City, Taiwan) and anti-GAPDH (Abcam Inc., Cambridge, UK) and were then incubated with alkaline phosphatase-conjugated secondary antibodies. The results were visualized by a colorimetric reaction using Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI, USA).
For Western blot analysis using E. coli, 0.5 ml of the culture medium (LB medium at 37°C, 12–16 h) was harvested and resuspended in 100 μl lysis buffer (1 mM EDTA, 1 mg/ml lysozyme) and incubated at room temperature for 15 min. Aliquots (10 μl) of the homogenate from each clone were resolved by 15% SDS-PAGE and subjected to Western blotting. A commercially available polyclonal antibody specific for the Ca²⁺ channel β₂ subunit (Sigma-Aldrich) was used, followed by a secondary anti-rabbit IgG antibody conjugated to alkaline-phosphatase (Promega, Madison, WI, USA).

Murakami M., Ohba T., Murakami A.M., Han C., Kuwasako K, & Itagaki S. (2019). A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells. PLoS ONE, 14(5), e0216169.

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Immunoblotting Analysis of Gravid Worms

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Gravid adult worms were transferred to tubes containing M9 buffer (22 mM KH₂PO₄, 42 mM Na₂HPO₄, 86 mM NaCl, and 1 mM MgSO₄•7H₂O), washed 4 times with M9 containing 0.1% Tween-20 and then resuspended on 1.5X sample buffer (87.5 mM Tris-HCl pH 6.8, 2.5% SDS, 150 mM DTT, 7.5% glycerol, bromophenol blue). After lysing by sonication and boiling, the equivalent of 8–12 worms was loaded onto 4–12% NuPAGE Bis-Tris Gels (Invitrogen). Proteins were then transferred to nitrocellulose membranes, probed with primary antibodies (see Key Resources Table) and detected using either horseradish (HRP)- conjugated secondary antibodies and WesternBright Sirius (Advansta) chemiluminescent substrate or, for the α-loading control, using alkaline phosphatase (AP)-conjugated secondary antibody and Western Blue® Stabilized Substrate for Alkaline Phosphatase (Promega). Membranes were imaged using a ChemiDoc MP imaging system (BioRad). For immunoblotting of U2OS samples, cells were collected by trypsinization, washed once with 1X PBS, resuspended on 1.5X sample buffer and boiled for 5 minutes before loading onto gels.

Lara-Gonzalez P., Moyle M.W., Budrewicz J., Mendoza-Lopez J., Oegema K, & Desai A. (2019). The G2-to-M transition is ensured by a dual mechanism that protects cyclin B from degradation by Cdc20-activated APC/C. Developmental cell, 51(3), 313-325.e10.

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Viral Envelope Protein Detection

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Purified RNA was transfected into BHK21 cells via electroporation, 3 μg per 200 μl of 10⁶ BHK21 cells. Fourteen μl of clarified supernatant was fractionated on 4–12% SDS-PAGE and blotted onto nitrocellulose filters (Invitrogen, CA). HIV Env bands were detected by monoclonal antibody (mAb) 16H3 (Gao et al., 2009 (link)) followed by goat anti-mouse IgG-HRP (Sigma, Cat# A8924-.5ML). Env bands were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific, Cat#. 32106). Yellow fever viral envelopes were fractionized in SDS-PAGE and detected by Dengue mAb 4G2 (Henchal et al., 1982 (link)) under non-reducing conditions and then conjugated with goat anti-mouse IgG-AP (Sigma, Cat # A3652-.5ML). Yellow fever viral envelope bands were developed with Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega Cat# S3841).

Yu J.S., Liao H.X., Pritchett J., Bowman C., Vivian C., Parks R., Xia S.M., Cooper M., Williams W.B., Bonsignori M., Reed S.G., Chen M., Vandergrift N., Rice C.M, & Haynes B.F. (2017). Development of a Recombinant Yellow Fever Vector Expressing a HIV Clade C Founder Envelope gp120. Journal of virological methods, 249, 85-93.

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Western Blot Analysis of His-Tagged Proteins

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For Western blot analysis, 12% SDS-PAGE was carried out to separate proteins prior to transfer onto a nitro-cellulose membrane (Pall) at 100 mA for 1 h using a Semi-Dry Trans-Blot Cell (Bio-Rad). Non-specific protein-protein interactions were blocked using 5% non-fat dry milk in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20) for 1 h. Membranes were incubated overnight at 4 °C with mouse anti-His antibody (Abmart, Shanghai) diluted 1:5,000 in blocking buffer, and washed three times for 5 min each time in TBST buffer. The second anti-body, AP-conjugated goat anti-mouse IgG (Promega) was diluted 1:7,500 in TBST buffer, incubated with the membrane for 1 h, and washed three times for 5 min each time in TBST buffer, followed by visualization with Western blue stabilized substrate for alkaline phosphatase (Promega).

Meng H., Wang Z., Meng X., Xie L, & Huang B. (2015). Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea. Genetics and Molecular Biology, 38(3), 381-389.

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Total RNA Extraction and qPCR Analysis

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Total RNA was extracted as described previously [19 (link)]. For reverse transcription (RT) quantitative real-time PCR (RT-qPCR), 1 μg of total RNA was first treated with DNase (Thermo Scientific) and then subjected to cDNA synthesis. The resulting cDNA was used for RT-qPCR with the appropriate primers (Table 1) and iTaq™ universal SYBR® Green Supermix (Bio-Rad). The raw Ct data were normalised against actin or EF1α mRNA.
Total soluble proteins were isolated from infiltrated leaf patches using extraction buffer (1 M Tris-HCl, pH 7.5, and 20 % glycerol). The homogenate was centrifuged (16,000 × g, 30 min, 4 °C) to remove cell debris, and the protein concentration was measured using a Bio-Rad Protein Assay (Bio-Rad). An equal amount (50 μg) of the proteins was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were electroblotted onto a PVDF filter (Roche), and GFP was probed using primary monoclonal antibody (anti-GFP monoclonal antibody, Thermo Scientific). The GFP protein was visualised using Western Blue^® Stabilized Substrate for Alkaline Phosphatase (Promega).

Wieczorek P, & Obrępalska-Stęplowska A. (2016). The N-terminal fragment of the tomato torrado virus RNA1-encoded polyprotein induces a hypersensitive response (HR)-like reaction in Nicotiana benthamiana. Archives of Virology, 161, 1849-1858.

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Western Blot Analysis of LuloHya Protein

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Western blot was carried out as described elsewhere [9 (link)]. Briefly, the content of 5 Lu. longipalpis SG and 100 ng of LuloHya were separated by NuPAGE protein gels. Proteins were transferred to a nitrocellulose membrane (iBlot, Invitrogene) that was blocked overnight at 4°C with blocking buffer: 50 mM Tris, 150 mM NaCl containing 5% (w/v) powdered nonfat blotting-grade milk (Bio-Rad) and 0.05% of Tween-20 (Sigma). Mouse anti-LuloHya antibodies were diluted in blocking buffer (1:5,000) and incubated for 90 min. Goat anti-mouse conjugated to alkaline phosphatase (Sigma) diluted in blocking buffer (1:10,000) was used as a secondary antibody. Immunogenic protein bands were visualized by Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI) and reaction was stopped with distilled water.

Martin-Martin I., Chagas A.C., Guimaraes-Costa A.B., Amo L., Oliveira F., Moore I.N., DeSouza-Vieira T.S., Sanchez E.E., Suntravat M., Valenzuela J.G., Ribeiro J.M, & Calvo E. (2018). Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection. PLoS Pathogens, 14(5), e1007006.

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Quantitative Dot Blot Analysis of Proteins

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The dot blot was performed as described previously.^{40 (link)} 2 ul of dissolved needles and soluble control were pipetted and dried onto a nitrocellulose membrane for 15 min. The membrane was blocked overnight with 5% powdered milk (Sigma-Aldrich) in TBS–0.1% Tween 20 (TBST) at 4°C overnight. The proteins were detected using 6x-His epitope tag primary antibodies raised in mice (ThermoFisher). After 2 hrs, the membrane was washed three times with TBST and incubated with goat anti-mouse secondary antibodies conjugated to alkaline phosphatase at 1:5000 dilution. The proteins were then detected using Western Blue Stabilized Substrate for alkaline phosphatase (Promega cat # S3841) and imaged after 5 min incubation.

Yenkoidiok-Douti L., Barillas-Mury C, & Jewell C.M. (2021). Design of Dissolvable Microneedles for Delivery of a Pfs47-based Malaria Transmission-blocking Vaccine. ACS biomaterials science & engineering, 7(5), 1854-1862.

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Western Blot Analysis of SIRT1 Protein

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Frozen tissues were homogenized before being lysed using T-PER™ Tissue Protein Extraction Reagent (ThermoFisher Scientific) containing protease inhibitor cocktail (Sigma Aldrich). Whole protein extracts were resolved by electrophoresis on 8% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), then electro-transferred onto polyvinylidene difluoride membranes (Immobilon-P, PVDF, 0.45 µm, Merck Millipore) in transfer buffer (25 mM Tris-HCL (pH 7.6), 192 mM glycine, 10% methanol). The membranes were blocked with 5% non-fat milk in 0.1% TBS-tween and later immunoblotted with monoclonal anti-SIRT1 antibody (1/500, MAb-063-050, Diagenode) or monoclonal anti-β-actin antibody (1/5000, CP01, Merck Millipore). Membranes were then washed and incubated with alkaline phosphatase-conjugated secondary antibody anti-mouse IgG (1/2000, S3721, Promega). Immunolabeling was detected using Western Blue^® Stabilized substrate for Alkaline Phosphatase (Promega) at room temperature.

Rifaï K., Judes G., Idrissou M., Daures M., Bignon Y.J., Penault-Llorca F, & Bernard-Gallon D. (2017). Dual SIRT1 expression patterns strongly suggests its bivalent role in human breast cancer. Oncotarget, 8(67), 110922-110930.

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SDS-PAGE and Western Blot Analysis

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Purified virus fractions (1–12) were diluted in 4x SDS-PAGE loading buffer (160 mM Tris, 6.4% SDS, 20% glycerol), loaded onto 10% or 12% separating SDS-polyacrylamide (Bio-Rad) gels and run in Tris-Glycine-SDS (TGS) running buffer at 90 v for 2–3 h with EPS 1001 power supply (General Electric). For western blots with 4G2, samples were not heated or reduced; for blots with 6F3-1, samples were heated; and for prM, samples were heated and reduced with β-mercaptoethanol. Gels were transferred to methanol-pretreated PVDF membranes (Bio-Rad) in transfer buffer (2.5 mM Tris, 19.2 mM glycine, 20% methanol) for 15–17 h at 30 v. Membranes were blocked for 1 h at room temperature with blocking buffer (5% milk in PBS-Tween 20). Membranes were incubated with 4G2 (10 μg/mL, 1 h), 6F3-1 (neat, 2 h), prM antibody (1:1,000, 2 h) in blocking buffer. After five washes with PBS-Tween 20, membranes were incubated 1 h with 1:1,000 dilution of appropriate secondary anti-mouse or anti-rabbit IgG-AP conjugated antibody. After washing, a 30-min incubation with Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI) allowed for visualization of viral antigens.

Clark K.B., Hsiao H.M., Bassit L., Crowe JE J.r., Schinazi R.F., Perng G.C, & Villinger F. (2016). Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells. Virology, 493, 162-172.

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Western Blot Protein Detection

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Recombinant proteins were boiled under reducing conditions in SDS-PAGE loading buffer, separated using 12% SDS-PAGE gels and transferred on nitrocellulose membranes. The membrane was blocked for 2 h with 5% non-fat dry milk in TBS–0.1% Tween 20 (TBST). The proteins were detected using 6×-His epitope tag primary antibodies (ThermoFisher cat # MA1-21315) or animal sera at 1:1000 dilutions. The membranes were then washed three times with TBST and incubated with goat anti-mouse or anti-rabbit secondary antibodies conjugated to alkaline phosphatase at 1:5000 dilution. The proteins were then detected using Western Blue Stabilized Substrate for alkaline phosphatase (Promega cat # S3841) after 5 min incubation.

Canepa G.E., Molina-Cruz A., Yenkoidiok-Douti L., Calvo E., Williams A.E., Burkhardt M., Peng F., Narum D., Boulanger M.J., Valenzuela J.G, & Barillas-Mury C. (2018). Antibody targeting of a specific region of Pfs47 blocks Plasmodium falciparum malaria transmission. NPJ Vaccines, 3, 26.

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