Spinal cords and ventral midbrains dissected from adult mice were kept frozen at −80°C. After thawing on ice and 3 rinses in PBS, tissues were resuspended in 500 μl dissociation buffer (DB: 50 mM Tris–HCl pH8.0, 0.3 M NaCl, Triton X‐100 1%, EDTA‐free Complete Proteases inhibitor cocktail from Roche and 250 U/ml of benzonase endonuclease from Sigma). After homogenization in a Dounce tissue homogenizer, 10 times with the A piston and 10 times with the B piston, lysates were incubated for 30 min on ice, sonicated for 5 min in Diagenode Bioruptor set with 30/30 s ON/OFF at high power, incubated on ice for 30 min, and centrifuged for 10 min at 12,000 g. Supernatants were harvested and their protein contents measured (Pierce BCA assay) and frozen at −80°C.
Complete proteases inhibitor cocktail
Manufactured by Roche
Sourced in Switzerland
The Complete Protease Inhibitor Cocktail is a ready-to-use solution designed to inhibit a broad spectrum of serine, cysteine, and metalloproteinases. It is formulated to provide effective protease inhibition in a wide range of applications, including cell and tissue lysate preparation, protein purification, and enzymatic assays.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor, by Diagenode (1 mentions)
Benzonase endonuclease, by Merck Group (1 mentions)
Coomassie blue, by Merck Group (1 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
V3 western workflow, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
P70s6k, by New England Biolabs (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
Appropriate secondary antibody, by Agilent Technologies (1 mentions)
Gapdh, by Merck Group (1 mentions)
7 protocols using complete proteases inhibitor cocktail
1
Tissue Homogenization and Protein Extraction
2
Insulin Receptor Activation Assay
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Cells were plated at a density of 3 × 105 cells in 60 mm diameter dishes and allow to equilibrate overnight. Full medium was replaced with serum-free medium (SFM) for 24 hours. Cells were then treated, washed twice with ice-cold PBS and lysed with lysis buffer of 50 mM Tris-Cl (pH 7.4), 1% Nonidet P-40, 2 mM EDTA (pH 8.0), 100 mM NaCl, 10 mM sodium orthovanadate, and with complete proteases inhibitor cocktail (Roche Diagnostics). Lysates were centrifuged at 12,000 g for 30 minutes at 4°C. Protein concentrations were measured using bicinchoninic acid protein assay reagent kit (Pierce). Whole cell lysates (50 μg) were boiled in 5X Laemmli loading buffer, separated by 8% SDS-PAGE, transferred to PVDF membrane and immunoblotted according to manufacturer guidelines. For Immunoprecipitation (IP), whole-cell lysates were incubated with either anti-InsR antibody or mIgG overnight at 4°C. Protein A/G PLUS-Agarose bead slurry was added into the samples and incubated for 4 hours at 4°C. Beads were washed with lysis buffer 5 times and boiled in 5X Laemmli loading buffer. Samples were resolved by 8% SDS-PAGE, transferred to PVDF membrane and immunoblotted.
3
Protein Extraction from E. histolytica
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Protein extraction was performed on 15,000 E. histolytica trophozoites grown in TYI-S-33 medium, incubated with 150 μM of kaempferol or 150 MTZ for 90 min at 37 °C, and then lysed in lysis buffer with complete proteases inhibitor cocktail (Roche, Basel, Switzerland); 100 mM PHMB (Sigma-Aldrich, MO, USA); 1 mM E64 (Thermo Scientific, MA, USA); 100 mM Tris (Sigma-Aldrich, MO, USA); 100 mM PMSF (Thermo Scientific, MA, USA); and 0.5 M iodoacetamide (Sigma-Aldrich, MO, USA). Three cycles of freezing and thawing were subsequently carried out, the supernatant was retrieved and centrifuged at 15,000× g for 5 min at 4 °C, the protein concentration was determined using Nanodrop Lite (Thermo scientific, MA, USA), and the protein integrity was determined by 10% SDS-PAGE and staining with Coomassie Blue (Sigma-Aldrich, MO, USA) [25 (link)].
4
Insulin Receptor Activation Assay
5
Western Blot Analysis of Lysed Cells
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Cell lysates of the OV-MZ-6-derived cell lines were generated using Tris-buffered saline containing 1% Triton X-100 and 0.1% complete proteases inhibitor cocktail (Roche). Protein concentrations were determined with the bicinchoninic acid protein assay (Thermo Fisher, Dreieich, Germany) and 40 µg/lane of protein was electrophoresed on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany) via semi-dry blotting. PVDF membranes were incubated with primary antibodies (Table S2 ) overnight at 4 °C and the secondary antibody (horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit IgG, 1:5000; Millipore) 1 h at room temperature. Proteins were visualised using enhanced chemiluminescence substrate (Thermo Fisher) with a V3 Western Workflow (Bio-Rad, Dreieich, Germany). For Coomassie Blue staining, SDS polyacrylamide gels were fixed in 50% methanol plus 10% acetic acid, stained with 0.1% Coomassie R-250 blue (Thermo Fisher) solution (40% ethanol plus 10% acetic acid) and destained in 40% methanol plus 10% acetic acid.
6
Quantification of NAMPT in HepG2 Cells
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0.3 × 106 HepG2 cells per well were seeded in 6 well plates and incubated as described above. Cells were harvested in modified RIPA buffer containing 50 mM TrisHCl pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 1× Roche complete proteases inhibitor cocktail; 1 mM EDTA; 1 mM sodium orthovanadate, 1 mM sodium fluoride, 5 mM nicotinamide, 5 μM Trichostatin A and 1× Roche Complete protease inhibitor cocktail. Protein amount of cell lysates was determined using BCA protein assay (Pierce, Thermo Scientific). 10 μg or 30 μg protein was separated by SDS-PAGE and semi-dry transferred to nitrocellulose membranes as described before [18 ]. For detection of NAMPT in the supernatant, 24 μl of samples were used and processed as described above.
7
Insulin-like Growth Factor-I Signaling Pathway
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LipPD1 cells were seeded at a density of 200,000 cells per well, incubated for 48 h and stimulated with 10 nM recombinant human insulin-like growth factor-I (hIGF-I) (Pharmacia Biotech) for 15 min. Cells were lysed with modified RIPA buffer (50 mM Tris HCl, pH 7.4; 1% NP40; 0.25% sodium deoxycholate; 1x Roche complete proteases inhibitor cocktail (Roche); 1 mM EDTA; 1 mM sodium orthovanadate; and 1 mM sodium fluoride). Immunoblots were performed to detect the amount of PTEN, phospho-AKT (Thr308), AKT, phospho-mTOR (Ser2448), mTOR, phospho-p70S6K (Thr389), and p70S6K (New England Biolabs). GAPDH (EMD Millipore) served as loading control. Appropriate secondary antibodies (Dako) were used. Protein bands were detected by Classico Luminata TM (Millipore) or Amersham TM ECL (GE Healthcare Life Sciences). Western blots were quantified using ImageJ densitometry software.
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