Cells were washed once with PBS and lysed with Triton X-100 lysis buffer (50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 2 mM dithiothreitol, 2 mM sodium orthovanadate) containing the protease inhibitor cocktail Complete™ (Roche Diagnostics, Mannheim, Germany). Cell lysates as cytoplasmic fractions were collected as supernatants by centrifugation (15,300g, 5 min). Precipitates were washed twice with Triton X-100 lysis buffer, and then solubilized as nuclear fractions. Proteins (30 μg/lane) were separated by SDS–polyacrylamide gel electrophoresis and transferred onto Hybond-ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were blocked with 4% skim milk in 0.5% Tween 20–PBS, and then reacted with primary antibodies and HRP-conjugated secondary antibodies (Jackson ImmunoResearch). Protein bands were developed with Western blotting detection reagent (GE Healthcare) and detected by exposure to Hyperfilm™ ECL (GE Healthcare) or ImageQuant LAS 4000 mini (GE Healthcare).
Cocktail complete
Manufactured by Roche
Sourced in Germany
Cocktail Complete is a laboratory instrument designed for the automated preparation of reagent mixtures, commonly known as 'cocktails'. It provides precise and consistent mixing of multiple liquid components to create solutions required for various analytical and experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay reagent, by Bio-Rad (2 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Western blotting detection reagent, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Nitrocellulose, by Thermo Fisher Scientific (1 mentions)
Gs 800 calibrated imaging densitometer, by Bio-Rad (1 mentions)
Cambridge gel doc system and software, by Uvitec (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Phostop, by Roche (1 mentions)
Nanog, by Santa Cruz Biotechnology (1 mentions)
Ps6 ser235 236, by Cell Signaling Technology (1 mentions)
P 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Atg5 atg12, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
7 protocols using cocktail complete
1
Nuclear and Cytoplasmic Protein Extraction
2
Immunoprecipitation and Immunoblotting of ZO-2
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Immunoprecipitation of ZO-2 was done using 1 μl of ZO-2 antibody (Cat. 71-1400; Invitrogen, Carlsbad, CA) per 300 μg of protein in the lysate of parental MDCK cells and following a protocol previously described (Raya-Sandino et al., 2017 ). The radioimmunoprecipitation assay buffer employed contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40 (vol/vol), and the protease inhibitor cocktail Complete (Cat. 11697498001; Roche Diagnostics, Mannheim, Germany). The blot for TEAD in the ZO-2 immunoprecipitate was done using the Tidy blot reagent (Cat. STAR209; BioRad, Hercules, CA) following the manufacturer’s instructions.
3
Protein Extraction from Cultured Cells
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Extraction of cellular proteins was performed as previously described [37 (link)]. In brief, following incubation of cells with the test compounds, cells were collected in cold PBS, pH 7.4 and centrifuged together with the cell culture medium at 4° C and 250 × g for 7 min. After one washing step with cold PBS, cells were lysed with 100 μl of RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium desoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS)) supplemented with the protease inhibitor cocktail Complete™ according to the manufacturer’s instructions (Roche Diagnostics, Boulogne-Billancourt, France). The cell lysate was left on ice for 15 min, subjected to sonification (3 × 1 min) at 4° C and then cell debris was removed by centrifugation at 16,250 × g at 4° C for 30 min. Nuclear and cytosolic proteins were also prepared as previously described [38 (link)]. The protein content of the supernatant was determined according to the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad, Marnes-la-Coquette, France).
4
Protein Extraction from Cell Pellets
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Pellets were lysed with 40–60 μl of RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium desoxycholate, 1% Triton X–100, 0.1% sodium dodecyl sulfate (SDS) supplemented with the protease inhibitor cocktail Complete™ according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany)). The cell lysate was left on ice for 30 min, before being subjected to sonification (3 × 30 s) at 4 °C. Then the cell debris was removed by centrifugation at 16100xg at 4 °C for 30 min. The protein content of the supernatant was determined according to the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad, Munich, Germany).
5
Western Blot Analysis of Proteins
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BC cells were lysed using lysis buffer (Hepes pH 7.4 20 mM, NaCl 150 mM, 10% Glycerol, 1% Triton X-100) with protease inhibitors cocktail Complete (Roche Applied Science, Penzberg, Germany) and sodium orthovanadate or Phostop (Roche). Proteins were resolved on SDS-polyacrylamide gel electrophoresis and blotted on nitrocellulose (Thermo Fisher Scientific) or PVDF (Merck Millipore, Darmstadt, Germany) membranes. Detection was performed with ECL Detection Reagent (GE Healthcare) according to manufacturer’s protocol. ECL signals were detected, recorded and measured by either the GS-800 Calibrated Imaging Densitometer and the Quantity One Software (BioRad, Hercules, CA) or the Li-Cor scanner and Image Studio software (LI-COR Biosciences Inc., Lincoln, NE, USA) or the Uvitec Cambridge gel doc system and software (Cambridge, UK).
6
Immunoblotting Analysis of Stemness and Autophagy
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For immunoblotting, cell lysates were obtained by incubating cells in RIPA buffer containing PBS solution, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS (Sigma), protease and phosphatase inhibitors (cocktail Complete, Roche), 5 mM EGTA and 10 mM β-glycerophosphate. Equal amounts of protein extracts were run on polyacrylamide gel electrophoresis, transferred to PVDF-FL membranes (Millipore) and blotted with primary antibodies according to the manufacturer’s recommendations. Horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse antibodies (Pierce) were used as secondary antibodies. Proteins on membranes were visualized by means of ECL (Amersham). The relative band intensity (normalized to α-tubulin) was quantitated using Image Lab version 6.0 (Bio-Rad).
Primary antibodies were as follows: Oct3/4 (#sc-5279), Sox2 (#sc-17320) and Nanog (#sc-376915) from Santa Cruz Biotechnology; α-tubulin (Sigma #T5168); GAPDH (#2118), H2AX (Ser139) (#9718), Klf4 (#4038), Atg5/Atg12 (#8540), BECLin-1 (#3738), AMPK (#5832), AMPK Thr172 (#2535), Ulk1 (#8054), ULk1 Ser555 (#5869), Ulk1 Ser757 (#14202), Ulk1 Ser317 (#12753), mTOR Ser2448 (#5536), pS6 Ser235/236 (#2211), p4EBP1 Thr37/46 (#2855), p70S6K Thr389 (#9206) (Cell Signaling); LC3 (#PM036) from MBL International; p62 (#610832) from BD Biosciences-US.
Primary antibodies were as follows: Oct3/4 (#sc-5279), Sox2 (#sc-17320) and Nanog (#sc-376915) from Santa Cruz Biotechnology; α-tubulin (Sigma #T5168); GAPDH (#2118), H2AX (Ser139) (#9718), Klf4 (#4038), Atg5/Atg12 (#8540), BECLin-1 (#3738), AMPK (#5832), AMPK Thr172 (#2535), Ulk1 (#8054), ULk1 Ser555 (#5869), Ulk1 Ser757 (#14202), Ulk1 Ser317 (#12753), mTOR Ser2448 (#5536), pS6 Ser235/236 (#2211), p4EBP1 Thr37/46 (#2855), p70S6K Thr389 (#9206) (Cell Signaling); LC3 (#PM036) from MBL International; p62 (#610832) from BD Biosciences-US.
7
Brain Tissue Homogenization and Aβ/Tau Extraction
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Before extraction of Aβ and tau from brain tissue, 10% (w/v) homogenates were prepared in tissue homogenization buffer (20 mM Tris base, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA) with 100 mM phenylmethylsulphonyl fluoride, protease inhibitors [protease inhibitors cocktail Complete (Roche Diagnostic GmbH, Mannheim, Germany) plus pepstatin A (Sigma-Aldrich Inc., St. Louis, MO)] and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4 and 0.5 mM okadaic acid) added immediately before homogenization, as we have previously published [18 ,19 (link)]. Subsequently, brain homogenates were aliquoted, frozen, and stored at −80°C until used for extraction of soluble and insoluble fractions of both Aβ and tau, and their biochemical analyses (ELISA, Western blot). All quantitative biochemical analyses were performed in brain homogenate fractions of the entire left hemisphere.
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