Total proteins were extracted with NP40 Cell Lysis Buffer (10 mmol/L Tris–HCl [pH 7.5], 150 mmol/L NaCl, 1% NP-40) (Life Technologies) with the addition of Halt Protease Inhibitor Single-Use Cocktail 100 x (Thermo Scientific). For Western blot analysis, 50 μg per line of the whole lysates were separated by electrophoresis on NuPAGE® precasted Gels (4%–12%) (Invitrogen, Life Technologies) and electro-transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer packs, Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). After protein transfer, the membranes were blotted with the primary antibodies (see Supplementary Methods for detailed information).
Halt protease inhibitor single use cocktail 100x
Manufactured by Thermo Fisher Scientific
Sourced in United States
Halt Protease Inhibitor Single-Use cocktail (100X) is a concentrated solution designed to inhibit a broad range of serine, cysteine, and metalloproteases. It can be added directly to protein samples to prevent proteolytic degradation.
Automatically generated - may contain errors
Lab products found in correlation
Novex bis tris sds acrylamide gels, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo mini nitrocellulose transfer pack, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Quantity one analyzing system, by Bio-Rad (1 mentions)
Mitochondria isolation kit for tissue, by Abcam (1 mentions)
Kimble dounce tissue grinder, by Merck Group (1 mentions)
4 protocols using halt protease inhibitor single use cocktail 100x
1
Protein Extraction and Western Blot Analysis
2
SDS-PAGE and Western Blotting Protocol
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SDS-PAGE and Western Blotting (WB) were performed according to standard protocols [73 (link)]. Briefly, cells were lysed in lysis buffer containing 15mM Tris/HCl pH7.5, 120mM NaCl, 25mM KCl, 1mM EDTA, 0.5% Triton X100, Halt Protease Inhibitor Single-Use cocktail (100X, Thermo Scientific). Whole lysate (50µg per lane) were separated using 4-12% Novex Bis-Tris SDS-acrylamide gels (Invitrogen), electro-transferred on Nitrocellulose membranes (Bio-Rad), and immunoblotted with the appropriate antibodies. The antibodies against the following proteins were used for the procedure: Histone H3 (D1H2), HIF-1-α (#3716), E-cadherin (clone 24E10), Phospho-Akt (Ser473), Akt (pan) (C67E7), Caspase-7 (#9492), Caspase-3 (#9662), Cleaved PARP (Asp214; D64E10) were obtained from Cell Signaling (Beverly, MA). SIRT1 from Serotec and VEGF-A (MAB293) from R&D systems; α-Tubulin (C-20), MMP2 (2C1), DDR1 (C-20), p21Cip1(sc-397), p27Kip1(sc-528), Bcl-2 (sc-7382), and all secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Chemiluminescence was detected using Pierce ECL Western Blotting Substrate (cat. 32109, Pierce, USA).
3
SDS-PAGE and Western Blotting Analysis
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SDS-PAGE and Western blotting were performed according to standard protocols. Briefly, cells were homogenized in lysis buffer containing 15 mM Tris/HCl pH 7.5, 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton X-100, and Halt Protease Inhibitor Single-Use cocktail (100X, Thermo Scientific, Waltham, MA, USA). Whole cell lysates (50 µg per line) from transfected cell lines were separated using 4–12% Novex Bis-Tris SDS-acrylamide gels (Invitrogen), electro-transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and immunoblotted with the p27Kip1 (SX53G8.5) mouse mAb (Cell Signaling, Beverly, MA, USA). Membranes were washed 3 times in PBS-Tween and then incubated with a secondary antibody conjugated with horseradish peroxidase in 0.5% milk for 2 hours at room temperature. Chemiluminescence was detected using Western Blotting Luminol Reagent (sc-2048, Santa Cruz, Dallas, TX, USA). Signal intensity was quantified with the Quantity One Analyzing System (Bio-Rad).
4
Isolation of Mitochondria from Mouse Brain
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Mitochondria were isolated from fresh brain tissue from both WT and NR2F1 heterozygous (Nr2f1-HET) mice with a Mitochondria Isolation Kit for tissue (ab110168, Abcam, Cambridge, MA, USA), according to the manufacturer's instructions. Briefly, the mouse brain tissue was washed twice with the washing buffer and then suspended in mitochondria isolation buffer to be homogenized with a 2 ml Kimble dounce tissue grinder (D8938-1SET, Sigma-Aldrich) and spun at 1000 g for 10 min at 4°C; the supernatant was then transferred to a new tube and centrifuged again at 12,000 g for 15 min at 4°C. Mitochondria pellets were resuspended twice using mitochondria isolation buffer containing protease inhibitor cocktail (Halt Protease Inhibitor Single-Use Cocktail 100X; 78430, Thermo Fisher Scientific). Final mitochondria isolates were then aliquoted, stored at −80°C and subsequently used for WB analysis.
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