Bcip nbt

Manufactured by Beyotime

Sourced in China

BCIP/NBT is a colorimetric substrate system used for the detection and visualization of alkaline phosphatase (AP) activity in various applications, such as Western blotting, immunohistochemistry, and in situ hybridization. It produces a dark-blue insoluble precipitate at the site of AP activity.

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Lab products found in correlation

Alkaline phosphatase quantification kit, by Nanjing Jiancheng (2 mentions) Megashortscript t7 high yield transcription kit, by Thermo Fisher Scientific (2 mentions) Anti digoxigenin ap fab fragment, by Roche (2 mentions) Qiaquick gel extraction kit, by Qiagen (2 mentions) Inverted microscope, by Zeiss (2 mentions) Fixative solution, by Solarbio (1 mentions) Cetylpyridinium chloride monohydrate, by Solarbio (1 mentions) L glycerophosphate, by Merck Group (1 mentions) Microplate reader, by PerkinElmer (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Alizarin red, by Solarbio (1 mentions) Alexa fluor 680 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Anti phospho p38 mapk antibody, by Cell Signaling Technology (1 mentions) Anti p38 mapk, by Cell Signaling Technology (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Lysis buffer, by Beyotime (1 mentions) Alp detection kit, by Beyotime (1 mentions) Sds lysis buffer, by Beyotime (1 mentions) Ab8416, by Abcam (1 mentions)

Close spelling variants of this product

BCIP/NBT ALP color development kit (96 citations) NBT/BCIP staining kit (50 citations) BCIP/NBT solution (20 citations) BCIP/NBT kit (17 citations) BCIP/NBT alkaline phosphatase staining kit (16 citations) BCIP/NBT ALP staining kit (13 citations) BCIP/NBT ALP kit (12 citations) BCIP/NBT Alkaline Phosphatase Kit (10 citations) BCIP/NBT Alkaline Phosphatase (ALP) Color Development Kit (5 citations) BCIP/NBT alkaline phosphatase color reagent kit (3 citations)

25 protocols using bcip nbt

Osteogenic Induction of PDLCs by H2O2 and LIPUS

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Osteogenic induction was applied to detect the osteogenic differentiation capacity of PDLCs induced by H₂O₂ in the presence or absence of LIPUS pretreatment. Cells were seeded at a density of 1 × 10⁵ cells per well in a 6-well culture plate and incubated. When the cells reached 80% confluency, the medium was replaced with stem osteogenic differentiation medium [a-MEM supplemented with 10% FBS, 50 µg/mL of ascorbic acid (Sigma), 5 mM L-glycerophosphate (Sigma), and 100 nM dexamethasone (Sigma) for 7-21 days depending on the experiment.
Alkaline phosphatase (ALP) staining was determined using NBT/BCIP (Beyotime) according to the manufacturer's protocol after 7 days of osteogenic induction. ALP activity was evaluated using an AKP analysis kit (Nanjing Jiancheng Bioengineering Institute, China) after 7 days, according to the manufacturer's instructions.
Calcium accumulation was detected by fixing with 4% fixative solution (Solarbio Co., Ltd., Beijing, China) and staining with 0.2% Alizarin Red (Solarbio) after 21 days of osteogenic induction. To quantify mineralization, cells stained with Alizarin Red were destained with 10% cetylpyridinium chloride monohydrate (Solarbio), following which the extracted stain was transferred to a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader (Perkin Elmer, USA).

Ying S., Tan M., Feng G., Kuang Y., Chen D., Li J, & Song J. (2020). Low-intensity Pulsed Ultrasound regulates alveolar bone homeostasis in experimental Periodontitis by diminishing Oxidative Stress. Theranostics, 10(21), 9789-9807.

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Quantification of p38 MAPK Activation

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Intestinal tissue was isolated and homogenized in lysis buffer (Beyotime Biotechnology, China) on ice. The homogenate was centrifuged (14 000 rpm, 15 min, 4°C) and the level of total supernatant protein was measured by BCA protein assay kit (Beyotime Biotechnology, China). The protein samples (20 μg) were boiled in Laemmle’s sampling buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels. Next, the proteins were transferred to a polyvinylidene difluoride membrane and blocked with Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20. They were then incubated with anti-p38 MAPK or the anti-phospho-p38 MAPK antibody (1: 1000; Cell Signaling Technology, Danvers, MA) for 16 h at 4°C, followed by incubation with Alexa Fluor 680-conjugated anti-rabbit IgG (Molecular Probes). After washing 5 times for 5 min each time, the membranes were visualized by NBT/BCIP (Beyotime Institute of Biotechnology, Nantong, China). The protein bands were scanned and quantified using ImageJ software version 1.34 s (Wayne Rasband National Institute of Health) using β-actin for normalization.

Wan Y.D., Zhu R.X., Bian Z.Z, & Pan X.T. (2019). Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 4609-4616.

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Osteogenic Differentiation of BM-MSCs

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After reaching 80% confluence, BM-MSCs at passage 3 were cultured in osteogenic induction medium (R&D, USA) for 7~14 days as different experiments needed. The medium was changed every 3 days. Alkaline phosphatase (ALP) staining and ALP activity were determined using NBT/BCIP (Beyotime, China) and ALP detection kit (Beyotime, China) according to the manufacturer's protocol.

Liu C., Zhang H., Tang X., Feng R., Yao G., Chen W., Li W., Liang J., Feng X, & Sun L. (2018). Mesenchymal Stem Cells Promote the Osteogenesis in Collagen-Induced Arthritic Mice through the Inhibition of TNF-α. Stem Cells International, 2018, 4069032.

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Protein Extraction and Western Blot Analysis

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Total protein was extracted from the frozen tissue samples using ultrasound homogenization with SDS lysis buffer (Beyotime Biotechnology, China). Lysates were cleared by centrifugation at 4 °C. The collected supernatants were measured for protein concentration using a BCA protein assay before being separated on 10 % SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with anti-PZ antibody (Abnova Taiwan, 1:2500) in PBS with 1 %BSA. The next day, membranes were washed and then incubated for 2 h at room temperature with ALP-conjugated secondary antibody (1:20,000). Protein signals were detected using NBT/BCIP (Beyotime Institute of Biotechnology) and analyzed with IPP software. The PZ/actin ratio was used for statistical analysis.

Wang H., Huang F., Pan X.Y., Guan Z.B., Zeng W.B., Li M.J, & Zhang R.H. (2016). Quantification of protein Z expression in lung adenocarcinoma tissues and cells. SpringerPlus, 5(1), 1046.

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Osteogenic Differentiation of Cells

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Cells were inoculated in six-well plates (3×10⁵ /well) and cultured in OIM upon sub-confluence. ALP staining was performed on day 4 of mineral induction with NBT/ BCIP (Beyotime), and images were taken. ALP activity assay was achieved by a kit from Nanjing Jiancheng, and data were calculated as instructed.
Cells were treated with 4% paraformaldehyde and 1% alizarin red solution (ARS; pH 4.2) on day 14. The mineral nodules were photographed and then dissolved in 10% cetylpyridinium chloride. Next, the absorbance was determined at 562 nm.

Ma L., Wang X., Liu H., Jiang C., Liao H., Xu S., Guo Y, & Cao Z. (2019). CXXC5 Mediates P. gingivalis-suppressed Cementoblast Functions Partially via MAPK Signaling Network. International Journal of Biological Sciences, 15(8), 1685-1695.

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Hypoxia-Induced Protein Expression Analysis

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After hypoxia treatment, total protein was extracted using a Western & IP cell lysis kit (Beyotime)containing1mM PMSF. Nuclear and cytoplasmic proteins were separated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions.Whole cell extracts were then separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidenedi oride (PVDF) membrane (Beyotime). Afterblocking, the membranes were incubated at 4 o C overnight with rabbit polyclonal Antibodies against Vacuolar H + ATPase (VMA21/V-ATPase, 1:1000 dilution,ab242099, Abcam, Cambridge, UK), TUBB1/β-tubulin (1:1000 dilution, Sigma,T7816), laminB(1:1000 dilution, Sigma, SAB1306342), TFEB(1:1000 dilution, SAB2107961, Sigma), LAMP 2 (1:1000 dilution, AF1036, Beyotime), JNK1 (1:1000 dilution, 3708, CST), Phospho-JNK1 (1:1000 dilution, 9255, CST), c-Jun (1:1000 dilution, 9165, CST), Phospho-c-Jun(1:1000 dilution, 9261, CST), DR 5 (1:500 dilution, ab8416,Abcam, Cambridge, UK),Caspase-8 (1:1000 dilution, 4790, CST) and β-actin(1:1000 dilution, sc-8432, Santa Cruz), Then the membranes were washed with TBST and incubated with horseradish peroxidase-linked secondary antibodies (ZDR-5306, ZDR-5307,Zhong Shan, Beijing, China). After being washed with TBST, immunoreactive bands were visualized using NBT/BCIP (Beyotime)as substrate.

Yin J., He J., Han F., Gao Z.q., Deng F., Liao W.g., Zhang M.j., Qiu H., Li Z.f., Zhang Q., & Ni B. (2021). V-ATPase Deficiency Aggravates Hypoxia-induced Spermatogenesis Reduction by Promoting Spermatocyte Apoptosis via the JNK/c-Jun Pathway in Mice.

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Polyclonal Antibody Production and Validation

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The polyclonal antibodies of GSTΩs were produced by immunizing the mice (Luye Pharma Inc., Yantai, China) with the recombinant proteins (Yang et al., 2010) . The serum of one mouse injected with PBS served as the negative control. Afterwards, the specificity of the antibodies was confirmed by a western blotting analysis. After the proteins were separated by SDS-PAGE, the gels were transferred to PVDF membranes. The membranes were then respectively incubated with polyclonal antibodies (1:1000 dilution) followed by a reaction with alkaline phosphatase (AP)-conjugated secondary goat anti-mouse IgG (1:3000 dilution, Beyotime, China). The western blotting was developed by NBT/BCIP (Beyotime, China) and terminated by washing the PVDF membranes with deionized distilled water.

Chen L., Wu H., Zhao J., Zhang W., Zhang L., Sun S., Yang D., Cheng B, & Wang Q. (2018). The role of GST omega in metabolism and detoxification of arsenic in clam Ruditapes philippinarum. Aquatic toxicology (Amsterdam, Netherlands), 204.

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Osteoblast Differentiation Assays

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MC3T3-E1 cells were seeded in 6-well plates and cultured in osteogenic induction medium with stimulation for 14 days. Alkaline phosphatase (ALP) staining was performed with a BCIP/NBT (nitro-blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate) alkaline phosphatase colour development kit (C3206, Beyotime, China) according to the manufacturer’s instructions. When the cells were induced for 28 days, Alizarin Red S (ARS) staining was performed to detect calcium deposits with modified Alizarin Red S stain kit for calcium (G3280, Solarbio, China). Stained plates were photographed using a digital camera. Images of stained cells were captured under a light microscope (BX41, Olympus, Japan) and five randomly selected fields (× 10 magnification) were photographed in each well, analysed by Image J software (version 1.53n, National Institutes of Health, USA) according to previous protocol [27 (link)–29 (link)].

Luo W., Yao C., Sun J., Zhang B., Chen H., Miao J, & Zhang Y. (2024). Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis. BMC Musculoskeletal Disorders, 25, 45.

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Quantifying Osteogenic Differentiation via ALP

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MC3T3-E1 cells were grown in a differentiation-inducing medium for 7 days, and ALP was measured as a phenotypic marker of bone formation. Cells were rinsed 3 times with PBS before fixation in 4% paraformaldehyde at 4°C for 30 min. After washing 3 times with PBS, the cells were incubated in 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT; Beyotime) staining solution for 30 min at room temperature in the dark. ALP-positive cells were counted under a microscope.

Xiong L., Liu Y., Zhu F., Lin J., Wen D., Wang Z., Bai J., Ge G., Xu C., Gu Y., Xu Y., Zhou J, & Geng D. (2019). Acetyl-11-keto-β-boswellic acid attenuates titanium particle-induced osteogenic inhibition via activation of the GSK-3β/β-catenin signaling pathway. Theranostics, 9(24), 7140-7155.

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Quantifying Alkaline Phosphatase in rMSCs

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ALP was assessed as a marker of bone formation. In brief, rMSCs were cultured for 7 days in osteogenic medium. Next, the cells were fixed in 4% paraformaldehyde for half an hour at 4 °C, and then prepared BCIP/NBT (Beyotime) working solution was added to completely cover the cells for incubation in the dark for 30 min. ALP activity was detected by an Alkaline Phosphatase quantification Kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Then, ALP activity was assessed at a wavelength of 520 nm (BioTek Instruments, lnc., USA).

Zheng K., Bai J., Li N., Li M., Sun H., Zhang W., Ge G., Liang X., Tao H., Xue Y., Hao Y., Zhu C., Xu Y, & Geng D. (2021). Protective effects of sirtuin 3 on titanium particle-induced osteogenic inhibition by regulating the NLRP3 inflammasome via the GSK-3β/β-catenin signalling pathway. Bioactive Materials, 6(10), 3343-3357.

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