Mouse antibodies against Myc-tag (M185-3L/M047-3, MBL, Nagoya, Japan), rabbit antibody against GFP-tag (D110008, BBI, Shanghai, China) and sheep antibody against Tubulin (ATN02, Cytoskeleton, Denver, United States) were subjected to Western blot according to the manufacturer’s protocol. For IP, HEK-293T cells transfected with the desired plasmids by using Attractene Transfection Reagent (301005, QIAGEN, Hilden, Germany), according to the fast-forward protocol, were lysed in IP buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF and Roche complete EDTA free protease inhibitor cocktail). After quantification by using BCA Protein Assay Kit (P0012, Beyotime, Beijing, China), lysates were precleared with Protein G Agarose (P2009, Beyotime, Beijing, China) and then immunoprecipitated with mouse anti-Myc antibodies. After washing five times, the precipitates were resuspended in the 5XSDS loading dye, boiled for 5 min, and run on a 12.5% SDS-PAGE gel followed by Western blot analysis. Immunoreactive bands were detected by Typhoon laser scanners (FLA-9500, GE, United States).
Atn02
Manufactured by Cytoskeleton
The ATN02 is a cell-based assay kit developed by Cytoskeleton. It is designed to quantify the polymerization state of actin, a key structural protein in the cytoskeleton of cells. The kit provides the necessary components and reagents to measure the relative amounts of F-actin (filamentous actin) and G-actin (globular actin) in cell lysates.
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Lab products found in correlation
2 protocols using atn02
1
Myc-tag Antibody Western Blot and IP
2
Immunofluorescence Staining of Plant Root Tip Cells
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Slides preparation of root tip cells and immunofluorescence staining procedure were carried out basically following a previously reported method [53] . Briefly, light-grown four days seedlings were used for root tip cells slides preparation. To visualize microtubules and gamma tubulins, a sheep polyclonal anti-tubulin antibody (ATN02; Cytoskeleton, Inc.) and a mouse monoclonal anti-g-tubulin antibody (clone G9 [48] ) were used at 1:500 dilution and at 1:2000 dilution, respectively. As the secondary antibody, Alexa 488labeled goat anti-mouse IgG (ab150113; Abcam) and Alexa 568-labeled donkey anti-sheep IgG (ab175712; Abcam) were used at 1:500 dilution. Image acquisition was performed using a Leica TCS-SP8 confocal microscope system (Leica).
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