Cell proteomes were denatured at 95 °C for 5 min and resolved by SDS-PAGE (10% acrylamide), transferred to nitrocellulose membrane (45 V for 120 min), and blocked by 5% milk in TBS-Tween. The primary antibodies used and dilutions are as follows: anti-FLAG (Rabbit, Sigma-Aldrich, F7425, 1:20,000) and anti-GAPDH (Mouse, Santa Cruz, SC-32233, 1:10,000). The secondary antibodies used and dilutions are as follows: HRP-labeled anti-rabbit (Goat, Santa Cruz, SC-2030, 1:5,000) and HRP-labeled anti-mouse (Goat, Santa Cruz, SC-2005, 1:5,000).
F7425
Manufactured by Santa Cruz Biotechnology
F7425 is a laboratory equipment product manufactured by Santa Cruz Biotechnology. It is designed for use in scientific research and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti flag m2 sepharose, by Merck Group (1 mentions)
Sc 7392 hrp, by Santa Cruz Biotechnology (1 mentions)
Ab104135, by Abcam (1 mentions)
Retiga r6 monochrome camera, by Zeiss (1 mentions)
Donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
5 protocols using f7425
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Cellular Proteins
3
Cell Culture and Protein Co-Immunoprecipitation
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Monkey COS-7 cells, human HEK293 and U2OS were grown in DMEM medium with 4,500 mg l−1 glucose supplemented with 10% bovine calf serum (Hyclone). HeLa cells were grown in Eagle's MEM medium supplemented with L -glutamine, sodium bicarbonate, sodium pyruvate and 10% bovine calf serum. Transient transfections were performed with Lipofectamine 2000 according to recommended protocols. Typically, a total of 5 μg plasmid DNA was used per 60-mm dish; lysates were harvested 18 h later in ice-cold lysis buffer (0.5 ml; 25 mM HEPES, pH 7.3, 100 mM KCl, 5 mM MgCl2, 20 mM β-glycerophosphate, 5% glycerol, 0.5% Triton X-100, 5 mM dithiothreitol, 0.5 mM phenylmethyl sulfonyl fluoride, 1 mM Na3VO4 and 1 × protease inhibitor cocktail (Roche)). To test co-immunoprecipitation of proteins, the lysates were clarified by centrifugation (14,000g) and the clarified lysates were incubated while rolling (2 h) with 20 μl M2 anti-Flag Sepharose (Sigma-Aldrich, A2220). Rabbit anti-Flag (Sigma-Aldrich, F7425) or horseradish peroxidase-coupled anti-HA (Santa Cruz Biotechnology, sc-7392 HRP, 1 μg ml−1) were used for western blot analysis.
4
Immunofluorescence Staining Protocol
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Cells were seeded on cover slide in 12-well plate and fixed with 4% paraformaldehyde in room temperature for 15 min. Afterward, cells were permeabilized with pre-cooled 0.5% TritonX-100 (dissolved in PBS) on ice for 5 min. After blocking with 1% bovine serum albumin, cells were incubated with primary antibodies at room temperature for 1 h. Primary antibodies used are anti-FLAG (Sigma #F7425), anti-Myc tag (Santa Cruz #SC-40), anti-BRD4 (CST #13440) and anti-USP1 (Proteintech #14346-1-AP). Slides were then incubated with secondary antibodies for 1 h at room temperature (Invitrogen #715-545-150, #711-585-152, and #711-545-152). Nuclei were counterstained with 1.5 μM 4′,6-diamidino-2-phenylindole for 5 min. Finally, cells were mounted onto a slide with mounting medium (Abcam #AB104135). Photos were taken with QImaging Retiga R6 Monochrome camera connected to Zeiss-A1 Axiovert A1 fluorescence microscopy equipped with 63 × oil object lens.
5
Immunofluorescence Assay for Nucleolar Localization of Dyskerin
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To assess localization of FLAG-dyskerin to the nucleolus, HEK293 cells expressing FLAG-dyskerin constructs were fixed with 4% formaldehyde-PBS for 10 minutes at room temperature. The fixing solution was removed and coverslips were briefly rinsed with PBS, followed by permeabilization of cells with 0.1% Triton X-100-PBS for 5 minutes at 4˚C. Permeabilized cells were then washed with PBS before blocking in 5% BSA-PBS for 1 hour at room temperature. Cells were probed for FLAG-dyskerin with rabbit anti-FLAG (Sigma-Aldrich F7425, 1:500) or mouse anti-dyskerin (Santa Cruz H-3, 1:25) in PBG (1% cold fish water gelatin, 0.5% bovine serum albumin (BSA), in PBS) overnight at 4˚C in a humidity chamber. In the case of assessing localization of only exogenous FLAG-tagged dyskerin, this was followed by probing with mouse anti-fibrillarin (monoclonal antibody 72B9 obtained from Dr. Kenneth Michael Pollard, 1:30) as a nucleolar marker, in PBG at 37˚C for 1 hour. Coverslips were washed with PBS and immunostained in PBG with secondary antibodies conjugated to fluorescein isothiocyanate (FITC) (donkey anti-mouse IgG; Jackson ImmunoResearch Lab, Inc.,
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