Alkaline phosphatase kit

Manufactured by Promega

Sourced in United Kingdom

The Alkaline Phosphatase Kit is a laboratory reagent used to detect the presence and quantity of the enzyme alkaline phosphatase in biological samples. Alkaline phosphatase is an enzyme involved in various cellular processes and is often used as a marker in diagnostic testing.

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Lab products found in correlation

Mouse anti myc, by Santa Cruz Biotechnology (1 mentions) Rat anti ha, by Roche (1 mentions) Mouse monoclonal anti gfp, by Roche (1 mentions) Mouse anti myc antibody, by Santa Cruz Biotechnology (1 mentions) Rabbit anti gfp antibody, by Abcam (1 mentions) Rat anti ha antibody, by Roche (1 mentions)

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Alkaline phosphatase (42 citations) Alkaline phosphatase detection kit (2 citations)

5 protocols using alkaline phosphatase kit

Western Blot Analysis of Myc-Tagged Proteins

Cited 2 times

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To detect Myc-PI3K or Myc-PIKfyve or Myc-PX2, Myc-PX1, PX3-Ty or PX4-3HA, FYVE1-3HA or ACP-YFP or PPP1-3HA or NtPX1-3Ty or NtPX2-3Ty proteins, parasite lysates were separated on 3–8%, 6%, 8% or 10% acrylamide gels dependent on their size. Upon transfer to nitrocellulose membranes, the blots were probed with appropriate antibodies in 5% non-fat milk powder in TNT buffer (50 mM Tris pH 8.0; 150 mM NaCl; 0.05% Tween20). The primary antibodies used and their respective dilutions were: rat anti-HA (Roche) at 1/300 (Morlon-Guyot et al., 2014 (link)), mouse anti-Myc (Santa Cruz Biotechnology) at 1/100, mouse anti-Ty at 1/1000 (Daher et al., 2012 (link)), and mouse monoclonal anti-GFP (Roche) at 1/200 (Sheiner et al., 2011 (link)). Bound secondary conjugated antibodies were visualized using either the ECL system (Amersham Corp) or using alkaline phosphatase kit according to manufacturer’s instructions (Promega).

Daher W., Morlon-Guyot J., Sheiner L., Lentini G., Berry L., Tawk L., Dubremetz J.F., Wengelnik K., Striepen B, & Lebrun M. (2014). Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii. Cellular microbiology, 17(4), 559-578.

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Immunoblotting of HA-tagged Toxoplasma proteins

Cited 1 time

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To detect TgArk2-3HA, TgArk3-3HA and TgArk3i-3HA proteins, parasite lysates were separated on 6% acrylamide gels. Upon transfer to nitrocellulose membranes, the blots were probed with anti-HA antibodies in 5% non-fat milk powder in TNT buffer (50 mM Tris pH 8.0; 150 mM NaCl; 0.05% Tween20). The rat anti-HA (Roche) antibody was used at the dilution of 1/300 (Morlon-Guyot et al., 2014 (link)). Bound secondary conjugated antibodies were visualized using the alkaline phosphatase kit according to manufacturer’s instructions (Promega).

Berry L., Chen C.T., Reininger L., Carvalho T.G., El Hajj H., Morlon-Guyot J., Bordat Y., Lebrun M., Gubbels M.J., Doerig C, & Daher W. (2016). The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence. Cellular microbiology, 18(8), 1106-1120.

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Western Blot Detection of Parasite Proteins

Cited 1 time

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To detect HA₃-TgArk1 or DD-Myc-TgArk1WT or DD- Myc-TgArk1D/A or SAG1, GFP or TgINCENP1-GFP or TgIN-CENP1-HA₃ or TgINCENP1i-HA₃ or Myc-TgIN- CENP2 proteins, parasite lysates or eluted proteins were fractionated on 12 and 10% acrylamide gels, respectively, prior to detection. Separated proteins were transferred to nitrocellulose membranes and probed with appropriate antibodies in 5% non-fat milk powder in TNT buffer (50 mM Tris pH 8.0; 150 mM NaCl; 0.05% Tween20). The primary antibodies used for detection and their respective dilutions were: rat anti-HA antibodies (Roche) at 1/300, mouse anti-Myc antibodies (SANTA CRUZ BIOTECHNOLOGY) at 1/100, rabbit polyclonal anti-TgSAG1 at 1/1000 [36 (link)], rabbit anti-GFP antibodies (abcam) at 1/1000. Bound secondary conjugated antibodies were visualized using either the ECL system (Amersham Corp) or using alkaline phosphatase kit according to manufacturer’s instructions (Promega).

Berry L., Chen C.T., Francia M.E., Guerin A., Graindorge A., Saliou J.M., Grandmougin M., Wein S., Bechara C., Morlon-Guyot J., Bordat Y., Gubbels M.J., Lebrun M., Dubremetz J.F, & Daher W. (2018). Toxoplasma gondii chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny. Cellular and molecular life sciences : CMLS, 75(23), 4417-4443.

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Osteogenic Differentiation of MSCs under Normoxia and Hypoxia

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For osteogenic differentiation, MSCs were plated in 6-wells at a density of 2 × 10⁴ cells/cm². In the case of differentiation in the osteogenic linage, DMEM was supplemented with 10 mM β-glycerophosphate, 10 nM dexamethasone, and 0.1 mM L-ascorbic acid-2-phosphate. The cells in normal-normoxia group and osteonecrosis-normoxia group were exposed to 20% oxygen under osteogenic differentiation conditions, and the cells in osteonecrosis-low oxygen treated group and normal-low oxygen treated group were exposed to 2% oxygen. Differentiation was terminated after 14 days and ALP staining was performed by using an alkaline phosphatase kit according to the manufacturer's instructions (Promega, Southampton, UK). The resulting blue, insoluble, granular dye deposit indicated sites of alkaline phosphatase activity. For Alizarin Red S staining, cells were fixed in 10% formalin for 30 minutes and stained with 2% Alizarin Red S solution for 30 minutes. Subsequently, cells were rinsed once with PBS at room temperature.

Fan L., Liu R., Li J., Shi Z., Dang X, & Wang K. (2015). Low Oxygen Tension Enhances Osteogenic Potential of Bone Marrow-Derived Mesenchymal Stem Cells with Osteonecrosis-Related Functional Impairment. Stem Cells International, 2015, 950312.

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Western Blot Analysis of Tachyzoites

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Freshly released tachyzoites were harvested, washed in PBS and resuspended directly in SDS sample buffer. Polyacrylamide gels were run under non-reducing conditions. Separated proteins were transferred onto nitrocellulose membranes using a semi-dry electroblotter and probed with primary antibodies diluted in 5% non-fat milk powder in TNT buffer (50 mM Tris pH 8.0; 150 mM NaCl; 0.05% Tween20). After several washes, the nitrocellulose membrane was incubated for 1h with secondary antibodies coupled to alkaline phosphatase. Bound secondary conjugated antibodies were visualized using an alkaline phosphatase kit according to manufacturer's instructions (Promega).

Lentini G., Kong-Hap M., El Hajj H., Francia M., Claudet C., Striepen B., Dubremetz J.F, & Lebrun M. (2014). Identification and characterization of Toxoplasma SIP, a conserved apicomplexan cytoskeleton protein involved in maintaining the shape, motility and virulence of the parasite. Cellular microbiology, 17(1), 62-78.

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