1 step nbt bcip

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 1-Step NBT/BCIP is a chromogenic substrate solution used for the detection of alkaline phosphatase enzyme activity in various immunoassay and histochemical applications. The product contains nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3'-indolyphosphate (BCIP), which react with the alkaline phosphatase enzyme to produce a distinctive blue-purple precipitate.

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Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions) Dulbecco phosphate buffered saline (dpbs), by BasalMedia (2 mentions) Akp streptavidin, by BD (2 mentions) Lymphoprep, by Axis-Shield (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Gentlemacs dissociator, by Miltenyi Biotec (2 mentions) Trap assay kit, by Beyotime (1 mentions) Alizarin red s, by Merck Group (1 mentions) Skimmed milk, by Agilent Technologies (1 mentions) Immun blot, by Bio-Rad (1 mentions) Immun star westernc chemiluminescent kit, by Bio-Rad (1 mentions) Image lab software, by Bio-Rad (1 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions) Chemidoc xrs imaging system, by Bio-Rad (1 mentions) Iblot system, by Thermo Fisher Scientific (1 mentions) Imagequant las 4000, by GE Healthcare (1 mentions) Nuclear extract kit, by Active Motif (1 mentions) Anti c myc antibody, by BioLegend (1 mentions) Anti mouse igg ap linked antibody, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

1-Step NBT/BCIP plus Suppressor NBT-BCIP solution (1-Step(tm) NBT/BCIP Substrate Solution 1-Step™ NBT/BCIP Substrate Solution 1-step TM NBT/BCIP reagents 1-Step NBT/BCIP solution 1‐Step™ NBT/BCIP Kit 1-Step NBT/BCIP plus Suppressor Solution 1-Step NBT/BCIP substrate NBT/BCIP

32 protocols using 1 step nbt bcip

Histochemical Staining of Cells

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For ALP staining, the cells were fixed with 4% PFA for 15 min at room temperature, followed by incubated with 1-Step NBT-BCIP (ThermoFisher Scientific) at room temperature for 5–15 min until the desired color developed.
For Alizarin Red S staining, the cells were fixed with 4% PFA for 15 min at room temperature, then incubated with 0.2% Alizarin Red S (Sigma) in the dark for 10 min at room temperature and rinsed in distilled water.
For Oil Red O staining, cells were fixed with 4% PFA. Then, the cells were rinsed with 60% isopropanol for 2 min, followed by stained with freshly prepared Oil Red O working solution for 15 min at room temperature. The cells were then rinsed with 60% isopropanol for 30 s. The nuclei were lightly stained with alum haematoxylin and rinsed in distilled water.
For TRAP staining, the cells were fixed with 4% PFA for 15 min at room temperature, followed by stained with Sigma Diagnostics™ Acid Phosphatase Kit (ThermoFisher Scientific) according to the manufacturer’s instruction. The activity of tartrate-resistant acid phosphatase (TRAP) was also detected by TRAP Assay Kit (Beyotime) after cell lysis.

Yu L., Xie M., Zhang F., Wan C, & Yao X. (2021). TM9SF4 is a novel regulator in lineage commitment of bone marrow mesenchymal stem cells to either osteoblasts or adipocytes. Stem Cell Research & Therapy, 12, 573.

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Western Blot Assay Protocol

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Western blot assays [36 (link)] were carried out using minigels [37 ] transferred to PVDF membranes (Immun-Blot^®, BioRad Laboratories Inc.) using a Trans-Blot^® Turbo™ Transfer System (BioRad Laboratories Inc.). Transfer conditions were 25 V during 30 minutes. Membranes were washed in Tris-buffered saline with 1% Tween 20 (TBST) and blocked with 5% skimmed milk (Sigma-Aldrich Co.) in TBST overnight. After blocking, membranes were washed 3 times with TBST and incubated for 2 hours with M22.8 hybridoma supernatant (1:10 dilution in TBST with 2,5% skimmed milk) at room temperature (RT). For colorimetric Western blotting assays, goat anti-mouse IgG antibodies conjugated to AP (Dako) diluted 1:1,000 in TBST with 2.5% skimmed milk were used as secondary antibodies (1.5 hours, RT). Goat anti-mouse IgG antibodies conjugated to horse rabbit peroxidase (HRP) (Dako) diluted 1:50,000 in TBST with 2.5% skimmed milk were used as secondary antibodies (1 hour, RT) for chemiluminescent Western blot assays. Colorimetric Western blot assays were revealed with 1-Step NBT/BCIP (Thermo Scientific) whereas the Immun-Star™ WesternC™ Chemiluminescent Kit (BioRad Laboratories Inc.) was selected for chemiluminescent assays. Protein bands were analyzed using the ChemiDoc XRS imaging system in conjunction with ImageLab software (BioRad Laboratories Inc.).

Calvo-Iglesias J., Pérez-Estévez D., Lorenzo-Abalde S., Sánchez-Correa B., Quiroga M.I., Fuentes J.M, & González-Fernández Á. (2016). Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization. PLoS ONE, 11(3), e0152210.

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Shikonin Inhibits TNF-α-Induced IκB-α Phosphorylation

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Human dermal fibroblasts were pretreated with 1 μmol/L shikonin or 0.1% DMSO for 2 h, and then stimulated with 50 ng/ml TNF-α for 30 min. Then, cytoplasmic proteins were extracted by nuclear extract kit (Active Motif, Carlsbad, California, USA) according to the manufacturer's instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5–20% gradient gel (Atto Co., Tokyo, Japan), and transferred onto nitrocellulose membrane by a semi-dry transfer method using iBlot® system (Invitrogen, Carlsbad, CA, USA). After blocked with 2% BSA for 1 h at room temperature, membranes were incubated with anti-IκB-α phospho Ser 32, 36 mAb (1:250 dilution), followed by incubation with alkaline phosphatase-conjugated anti-mouse IgG pAb (1:2000 dilution) or HRP-DirecT-conjugated anti-GAPDH mAb (1:1000 dilution). Reactions were visualized using 1-step™ NBT/BCIP (Thermo Scientific, Rockford, IL, USA) or HRP detection systems (Atto Co., Tokyo, Japan). Specific bands were scanned and analyzed by ImageQuant Las 4000 (GE Healthcare Life Sciences, Uppsala, Sweden).

Yan Y., Furumura M., Gouya T., Iwanaga A., Teye K., Numata S., Karashima T., Li X.G, & Hashimoto T. (2015). Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro. Chinese Medical Journal, 128(16), 2228-2233.

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Detecting Chlamydial and Flagellar Proteins

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Western blot was performed as previously described.24 Chlamydial proteins were co-expressed with the c-Myc tag under the same inducible promoter. Thus, the detection of c-Myc was inferred as foreign chlamydial protein expression. The membrane was probed with 1:500 anti-c-Myc antibody (BioLegend, San Diego, CA, USA) and 1:1,000 anti-mouse IgG-AP-linked antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) as a secondary antibody. The intensity of the specific bands was visualized with 1-step NBT/BCIP (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) substrate solution (Thermo Fisher Scientific).
Western blot was also performed to detect the presence of flagellar proteins in the bacterial samples. The membrane was probed with 1:1,000 anti-E. coli Flagella H Pool A serum (Statens Serum Institut, Copenhagen, Denmark) as the primary antibody and 1:1,000 anti-rabbit IgG-AP antibody in TBST (Sigma-Aldrich, St Louis, MO, USA) as the secondary antibody. The intensity of the specific bands was visualized with a 1-step NBT/BCIP substrate solution (Thermo Fisher Scientific).

Montanaro J., Inic-Kanada A., Ladurner A., Stein E., Belij S., Bintner N., Schlacher S., Schuerer N., Mayr U.B., Lubitz W., Leisch N, & Barisani-Asenbauer T. (2015). Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface. Drug Design, Development and Therapy, 9, 3741-3754.

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Alkaline Phosphatase Activity Assay

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After 48 h of culture, cells were fixed with 3.7% formaldehyde (FA) solution (Sigma Aldrich, 252549-100ML) for 15 min at room temperature. Alkaline phosphatase assay was performed using 1-Step NBT/BCIP (Thermo Fisher Scientific, 34042) according to the manufacturer’s instructions.

Scaramuzzino L., Lucchino V., Scalise S., Lo Conte M., Zannino C., Sacco A., Biamonte F., Parrotta E.I., Costanzo F.S, & Cuda G. (2021). Uncovering the Metabolic and Stress Responses of Human Embryonic Stem Cells to FTH1 Gene Silencing. Cells, 10(9), 2431.

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Influenza A (H5N8) Virus Protein Analysis

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Electrophoretic separation of proteins was carried out using Laemmli PAGE in a discontinuous buffer system in the presence of 0.1% SDS in Tris-glycine buffer. Then, the proteins of cell lysates and culture medium, separated electrophoretically in a polyacrylamide gel, were transferred to a nitrocellulose membrane (Cytiva, Marlborough, MA, USA). Protein transfer was controlled by staining the membrane with crimson C for several minutes, followed by washing the dye with distilled water.
Western blot analysis was performed using the SNAP i.d system 2.0 (Merck Millipore, Burlington, MA, USA) in accordance with the manufacturer’s recommendations. The serum of a ferret immunized with influenza A (H5N8) virus (1:200 dilution) was used as primary antibodies (FBRI SRC VB «Vector», Rospotrebnadzor). A fraction of anti-ferret immunoglobulins from mouse serum (dilution 1:3000) (FBRI SRC VB «Vector», Rospotrebnadzor) was used as secondary antibodies. Immune complexes were visualized using anti-mouse antibodies conjugated to alkaline phosphatase (1:5000) (Sigma-Aldrich, Saint Louis, MO, USA) and subsequent incubation with phosphatase substrate 1-Step™ NBT/BCIP (Thermo Fisher Scientific, USA).

Litvinova V.R., Rudometov A.P., Rudometova N.B., Kisakov D.N., Borgoyakova M.B., Kisakova L.A., Starostina E.V., Fando A.A., Yakovlev V.A., Tigeeva E.V., Ivanova K.I., Gudymo A.S., Ilyicheva T.N., Marchenko V.Y., Sergeev A.A., Ilyichev A.A, & Karpenko L.I. (2024). DNA Vaccine Encoding a Modified Hemagglutinin Trimer of Avian Influenza A Virus H5N8 Protects Mice from Viral Challenge. Vaccines, 12(5), 538.

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Enumeration of IgG-secreting Cells

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Ninety-six-well multiscreen filtration plates (Millipore) were soaked with 30% ethanol and then rinsed and coated overnight at 4°C with 5 µg/ml polyclonal goat anti-mouse IgM+IgG+IgA (Millipore). The following day, the plates were washed and blocked with PBS--3% bovine serum albumin for 2 h at 37°C. Serial dilutions of single-cell suspensions of bone marrow were cultured on plates for 3 h at 37°C in medium prepared as described above. Cells were washed off, and the plates incubated with anti-mouse IgG-alkaline phosphatase (SouthernBiotech) for 1 h at 37°C. Finally, plates were washed with water before detection of IgG⁺ spots with 1-Step NBT-BCIP (nitro-blue tetrazolium chloride--5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) substrate (Thermo Scientific).

Gengenbacher M., Vogelzang A., Schuerer S., Lazar D., Kaiser P, & Kaufmann S.H. (2014). Dietary Pyridoxine Controls Efficacy of Vitamin B6-Auxotrophic Tuberculosis Vaccine Bacillus Calmette-Guérin ΔureC::hly Δpdx1 in Mice. mBio, 5(3), e01262-14.

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Alkaline Phosphatase Staining of hiPSCs

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hiPSCs were washed with PBS and fixed with 4% paraformaldehyde (PFA) solution and then rinsed with PBS three times. AP staining was performed using 1‐Step NBT/BCIP (Thermo Fisher Scientific) until the desired colour develops. The reaction was stopped by rinsing well in phosphate‐buffered saline (PBS).

De Angelis M.T., Santamaria G., Parrotta E.I., Scalise S., Lo Conte M., Gasparini S., Ferlazzo E., Aguglia U., Ciampi C., Sgura A, & Cuda G. (2019). Establishment and characterization of induced pluripotent stem cells (iPSCs) from central nervous system lupus erythematosus. Journal of Cellular and Molecular Medicine, 23(11), 7382-7394.

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Quantitative Analysis of mTOR Phosphorylation

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Cells were isolated, suspended in 1 × RIPA lyses buffer (Sigma) supplemented with Complete Protease Inhibitor tablets (1 per 50 ml) (Roche) and PhosSTOP phosphatase inhibitor tablets (1 per 10 ml), sonicated and then centrifuged at 14 000 g for 30 min at 4 °C. Seventy microgram of total protein was separated on 4–20% SDS-polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 4% bovine serum albumin (Sigma) in 1.0% Tween (Sigma) in PBS and probed with primary antibodies against mTOR pSer2448 (1:10 000; Abcam) and glyceraldehyde 3-phosphate dehydrogenase (1:10 000; Ambion, Life Technologies) overnight at 4 °C, then with alkaline phosphatase-conjugated secondary antibodies (Sigma) for 2 h at room temperature. The membrane was visualized using 1-step NBT/BCIP (Thermo Fisher Scientific). Imaging was done using a Bio-Rad Gel Doc Imaging System; the membrane was cut during imaging. Semi-quantitative analysis was done with NIH ImageJ (http://imagej.nih.gov/ij/), which was used to compare the density (intensity) of bands.

Topol A., English J.A., Flaherty E., Rajarajan P., Hartley B.J., Gupta S., Desland F., Zhu S., Goff T., Friedman L., Rapoport J., Felsenfeld D., Cagney G., Mackay-Sim A., Savas J.N., Aronow B., Fang G., Zhang B., Cotter D, & Brennand K.J. (2015). Increased abundance of translation machinery in stem cell–derived neural progenitor cells from four schizophrenia patients. Translational Psychiatry, 5(10), e662-.

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Immunoblotting of Ashbya gossypii Transformants

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A. gossypii transformants were grown on AFM containing 200 μM G418 at 28°C for 7 days. Protein expression was verified by harvesting the mycelium and preparing whole-cell protein extracts of the A. gossypii transformant colonies, as described previously (28 (link)). The prepared extracts were analyzed by immunoblotting using specific antisera (1:2,500 dilutions) for detection of the various FBDPs, except for the extracts containing tamavidin 1, where the blotted extract proteins were probed for bound biotin using the Vectastain ABC alkaline phosphatase system (Vector Laboratories) in combination with a 1-Step NBT/BCIP (nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyl phosphate) solution (Thermo Scientific). Antisera against Coprinopsis cinerea galectin 2 (CGL2) and Coprinopsis cinerea lectin 2 (CCL2) were described previously (18 (link), 28 (link)). The rabbit antisera against Marasmius oreades agglutinin (MOA), Aleuria aurantia lectin (AAL) and Sordaria macrospora transcript associated with perithecial development 1 (TAP1) were raised against the purified recombinant proteins by Pineda Antikörper-Service (Berlin, Germany). Expression and purification of these proteins from E. coli were carried out as previously described (28 (link), 35 (link), 55 (link)).

Tayyrov A., Schmieder S.S., Bleuler-Martinez S., Plaza D.F, & Künzler M. (2018). Toxicity of Potential Fungal Defense Proteins towards the Fungivorous Nematodes Aphelenchus avenae and Bursaphelenchus okinawaensis. Applied and Environmental Microbiology, 84(23), e02051-18.

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