Alkaline phosphatase conjugated secondary antibody

Manufactured by Cell Signaling Technology

Sourced in United States

Alkaline phosphatase-conjugated secondary antibody is a laboratory reagent used in various immunoassays and detection techniques. It serves as a detection tool, binding to a primary antibody and producing a colorimetric or chemiluminescent signal when exposed to a suitable substrate.

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Lab products found in correlation

β actin, by Abcam (1 mentions) Bca protein quantitation assay, by Keygen Biotech (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Polyvinylidene difluoride membranes, by Fujifilm (1 mentions) Rapid cbb kanto, by Kanto Chemical (1 mentions) Handy sonic ur 20p, by TOMY (1 mentions) E cadherin antibody, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions) Rabbit anti mmp 2, by Cell Signaling Technology (1 mentions) Mouse anti β actin, by Boster Bio (1 mentions) Rabbit anti rap2b, by Abcam (1 mentions)

Spelling variants of this product

Goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (3 citations) Alkaline phosphatase (2 citations)

6 protocols using alkaline phosphatase conjugated secondary antibody

Quantitative Protein Analysis by WB

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OC cells were collected and the protein concentrations were measured using the BCA Protein Quantitation Assay (Nanjing KeyGen Biotech Co., Ltd., Nanjing, Jiangsu, China). SDS-PAGE was used to separate the total protein and proteins were then transferred to PVDF membranes. PVDF membranes were blocked in 5% non-fat dry milk and probed with primary antibodies overnight at 4°C. PVDF membranes were incubated with alkaline phosphatase-conjugated secondary antibody and finally detected by chemiluminescence. Primary antibodies against KCNH3, RPA1, RPA2, and β-actin were obtained from Abcam (Cambridge, MA, USA). The alkaline phosphatase-conjugated secondary antibody was purchased from Cell Signaling Technology (Beverly, MA, USA).

Li Z., Huang L., Wei L., Zhang B., Zhong S., Ou Y., Wen C, & Huang S. (2020). KCNH3 Predicts Poor Prognosis and Promotes Progression in Ovarian Cancer. OncoTargets and therapy, 13, 10323-10333.

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LPS-Induced MAPK Activation Analysis

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For analysis of MAPK activity, the samples of tissue lysates were prepared from livers and kidneys of WT and SR-B transgenic mice (both PBS- and LPS-treated, n=3) sacrificed at 4 h after LPS injection (1 mg/kg, intraperitoneal). This time point was selected from additional experiments where the time dependent activation of MAPKs in mouse tissues following LPS injection was assessed. The tissue lysates were mixed with 2× SDS sample buffer and loaded onto 4–20% Tris-glycine SDS/PAGE gels. After the transfer, the membranes were probed with either one of three anti-phospho-MAPK antibodies or corresponding antibodies that recognize both active and inactive forms of each subfamily of MAP kinases, according to the manufacturer’s protocols. The MAPK antibodies used in this study included anti-phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) antibody, anti-ERK1/2 antibody, anti-phospho-SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵) antibody, anti-SAPK/JNK antibody, anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) antibody and anti-p38 MAPK antibody. The immunoreactive bands were detected using an alkaline phosphatase-conjugated secondary antibody (Cell Signaling Technology) and chromogenic substrate for alkaline phosphatase (Invitrogen).

Baranova I.N., Souza A.C., Bocharov A.V., Vishnyakova T.G., Hu X., Vaisman B.L., Amar M.J., Chen Z., Kost Y., Remaley A.T., Patterson A.P., Yuen P.S., Star R.A, & Eggerman T.L. (2016). Human SR-BI and SR-BII potentiate LPS-induced inflammation and acute liver and kidney injury in mice. Journal of immunology (Baltimore, Md. : 1950), 196(7), 3135-3147.

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Western Blot Analysis of Mouse Proteins

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Mouse tissues or cultured cells were lysed using lysis buffer (125 mmol/l NaCl, 20 mmol/l Tris–HCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate; pH 7.4) followed by centrifugation at 15,000 rpm for 5 min at 4 °C to remove undissolved debris. The lysates were then sonicated (Handy Sonic UR-20 P; Tomy Seiko Co, Ltd) on ice. Protein concentrations of the lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were loaded onto SDS–PAGE gels, transferred to polyvinylidene difluoride membranes (Wako), and incubated overnight with the primary antibody of interest. Alkaline phosphatase–conjugated secondary antibody (Cell Signaling) and 5-bromo-4-chloro-3-indolyl-phosphate–nitro blue tetrazolium (Wako) were used to detect the target proteins. The primary antibodies used for immunoblotting are listed in Table S2. Coomassie brilliant blue staining was conducted using Rapid CBB KANTO (KANTO Chemical Co) according to the manufacturer’s instructions.

Akasu R., Miyazaki T., Elhussiny M.Z., Sugiura Y., Tomitsuka Y., Haraguchi S., Otsu K., Chowdhury V.S, & Miyazaki A. (2022). Calpain-mediated proteolytic production of free amino acids in vascular endothelial cells augments obesity-induced hepatic steatosis. The Journal of Biological Chemistry, 298(6), 101953.

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Analyzing E-cadherin Expression in ESCs

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ESCs were treated with PDFV and the control peptide (10 μM) for 48 h. Then, cell extracts (40 μg) were obtained and resolved by 10% reducing SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked for 2 h at room temperature with a 3% bovine serum albumin (BSA) solution in 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.02% Tween 20 (TBST), and the membranes were incubated overnight at 4°C in a 1:500 dilution of E-cadherin antibody (Cell Signaling Technology, Danvers, MA, USA) and a 1:1000 dilution of β-actin (Cell Signaling Technology) in TBST containing 0.2% BSA. The membranes were washed five times with TBST and then incubated with alkaline phosphatase-conjugated secondary antibody (1:10, 000 dilution; Cell Signaling Technology). The bands were visualized using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution.

Xue Y., Xu P., Xu S., Xue K., Xu L., Chen J., Xu J., Shi X., Li Q, & Gu L. (2018). Peptidomic Analysis of Endometrial Tissue from Patients with Ovarian Endometriosis. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 47(1).

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Western Blot Analysis of Transfected Proteins

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HEK cells transfected with indicated constructs were directly lysed in Laemmli sample buffer 48 h after transfection. Samples were boiled and run on a 4–20% gradient sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel. Proteins were blotted on a nitrocellulose membrane and blocked with 5% (w/v) milk, 1% (v/v) Tween-20 in phosphate-buffered saline (PBS). Membranes were incubated with mouse anti-pan-Shank (UC Davis/NIH Neuromab Facility, clone N23B/4; 1 : 5000) and rabbit anti-GFP (eBiosciences; 1 : 1000) overnight at 4 °C, washed with PBS with 1% (v/v) Tween and incubated with alkaline phosphatase-conjugated secondary antibodies (Cell Signaling; 1 : 2000).

MacGillavry H.D., Kerr J.M., Kassner J., Frost N.A, & Blanpied T.A. (2015). Shank–cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses. The European journal of neuroscience, 43(2), 179-193.

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Quantifying Rap2B Protein Levels

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To determine Rap2B protein levels by western blot, cells were transiently transfected with plasmids for 24 h. Then, cells were harvested and washed twice with PBS. After cell lysis, the protein concentration was determined by using the bicinchoninic acid (BCA) assay. Proteins were separated by SDS-PAGE and then electrotransfer onto nitrocellulose membranes. The primary antibodies were utilized for western blot: rabbit anti-Rap2B (Abcam, Cambridge, MA, USA), rabbit anti-MMP-2 (Cell Signaling Technology, Beverly, MA, USA) and mouse anti-β-actin (Boster Biotechnology, Wuhan, China). The membranes were then washed and incubated with alkaline phosphatase conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) for 2 h at room temperature. Detection was performed by an enhanced chemiluminescence method.

Di J.H., Qu D.B., Lu Z., Li L.T., Cheng Q., Xin Y., Zhang L.Z., Zhang Y, & Zheng J.N. (2014). Rap2B promotes migration and invasion of human suprarenal epithelioma. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 35(9), 9387-9394.

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