One-hundred and fifty μg of protein per treatment were mixed with 2ug of primary antibody, in a dilution buffer —NaCl (Tris HCl pH 7.4, EDTA 10 nM, NP40 1%, NaF 50 mM, PMSF 1 mM, and protease inhibitors cocktail (Pierce) (Thermo Fisher Scientific) 1:100), and the samples were incubated at 4 °C for 4 h. Later, the protein mix was incubated with the washed resin (Protein G) (Thermo-Fisher Scientific) at 4 °C overnight. The next day, the samples were washed with a +NaCl wash buffer (Tris HCl pH 7.4 1 M, NaCl, EDTA 10 mM, NP40 1%, NaF 50 mM, PMSF 1 mM and protease inhibitors cocktail (Pierce) (Thermo Fisher Scientific) 1:100) and were reduced with 35 μl of a 1x loading buffer and boiling for 5 min. Subsequently, the immunoprecipitated complexes were subjected to electrophoresis and western blotting with primary antibodies against the corresponding essential nodes from intrinsic apoptosis pathway, the detection of the proteins in the complex was conducted as previously described and densitometry analyses were performed using ImageJ software [37] (link).
Protease inhibitors cocktail
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protease inhibitor cocktails are formulations designed to inhibit the activity of various proteases, which are enzymes that break down proteins. These cocktails contain a mixture of different protease inhibitors, each targeting specific types of proteases. The primary function of protease inhibitor cocktails is to preserve the integrity of protein samples by preventing unwanted proteolysis during protein extraction, purification, and analysis procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (7 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Uracil, by Merck Group (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Hrp substrate, by Merck Group (2 mentions)
Anti glud1 or anti gls1 antibodies, by Abcam (2 mentions)
Anti α actin mab, by Santa Cruz Biotechnology (2 mentions)
P11 phosphocellulose column, by Cytiva (2 mentions)
Whatman, by Cytiva (2 mentions)
M per lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Avantor (2 mentions)
Elisa quantikine r kits, by R&D Systems (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Ecl solution, by Cytiva (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Skim milk, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
90 ti rotor, by Beckman Coulter (1 mentions)
45 protocols using protease inhibitors cocktail
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of Glutamate Metabolism
3
Breast Tissue Fractionation for Proteomics
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Breast tissue at fifth, sixth, and seventh week was homogenized in PBS pH 7.4 in the presence of a protease inhibitor. The homogenate was centrifuged at 14 000 G for 15 min. The supernatant was transferred to a polycarbonate tube and centrifuged in a 90Ti rotor (Beckman, Brea, CA, USA) at 100,000 G for 1 h at 4 °C. The pellet was resuspended in lysis buffer for 2D Electrophoresis: 6 M urea, 50 mMDTT, 2% CHAPS in the presence of a protease inhibitors cocktail (Thermo Scientific, Waltham, MA, USA).
4
Expression and Purification of LTopIB
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Expression and purification of LTopIB was carried out as described previously (Villa et al., 2003 (link)). Briefly, LTopIB was purified from yeast strain EKY3 deficient in TopIB activity (MATα, ura3-52, his3Δ200, leu2Δ1, trp1Δ63, top1Δ:TRP1 ), that carries the bicistronic expression vector pESC-URA containing both subunits of LTopIB. Yeast were grown in yeast synthetic drop-out medium without uracil (Sigma) supplemented with 2% raffinose (w/v) to OD600: 0.8–1 and induced for 10 h with 2% galactose (w/v). Cells were harvested, washed with cold TEEG buffer (50 mM Tris–HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 10% glycerol) and resuspended in 15 mL of 1 x TEEG buffer supplemented with 0.2 M KCl and a protease inhibitors cocktail (Thermo Scientific). Protein extract, obtained lysing yeast cells, was loaded on a 5 mL P-11 phosphocellulose column (Whatman International Ltd. England). LTopIB protein was eluted at 4 °C with a discontinuous gradient of KCl (0.2, 0.4, 0.6 M) in TEEG buffer. The fractions eluted with 0.6 M KCl in TEEG buffer were supplemented with 50% glycerol and stored at −20 °C prior to using in enzyme assays.
5
Protein Isolation from Mammalian Cells
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For protein isolation, cells were washed with ice-cold PBS, next harvested and lysed with ice-cold RIPA buffer (ThermoFischer, Waltham, MA, USA), supplemented with protease inhibitors cocktail (Halt, ThermoFischer, Waltham, MA, USA) and centrifuged at 14,000× g for 15 min at 4 °C. The supernatants were stored at −20 °C until further use. Protein concentration was measured with a BCA kit (ThermoFischer, Waltham, MA, USA) following the manufacturer’s protocol.
6
WES Protein Analysis of Cell Lysates
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The protein levels in cells were determined by WES protein analysis (ProteinSimple, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, cells were washed with ice-cold PBS and lysed in MPER lysis buffer (Thermo Fischer Scientific) containing protease inhibitors cocktail (Thermo Fischer Scientific) by incubating for 15 min on ice. The cell debris was removed by centrifugation at 10,000× g for 15 min at 4 °C. The protein levels in clear cell lysate were measured by Bradford assay (VWR Life Science). Total protein (250 or 500 ng) was separated and immunoprobed in a capillary system using the WES ProteinSimple system: rabbit polyclonal antibody for mCherry (Abcam ab183628) and rabbit monoclonal antibody for GAPDH (Cell Signaling #5174) were used at 1:50 or 1:100 dilutions. Quantitative results such as molecular weight and signal intensity (area) were obtained using the WES ProteinSimple system software according to the manufacturer’s instructions.
7
Sam68 Protein Interactome Profiling
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P35–45 old Sam68 KO or WT mice were euthanized using CO2 and cortex was quickly dissected and Dounce homogenized 20 times in lysis buffer: 25 mM Tris pH 7.4, 50 mM KCl, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40 and 100 U/ml RNase inhibitor containing protease inhibitors cocktail (Thermo scientific). Lysates were incubated at 4°C with rocking for 1 hr and then centrifuged at 20,000 × g for 30 min. 1 mg of WT or Sam68 KO lysate was incubated with 2 mg of Sam68 antibody overnight at 4C and immunocomplexes recovered with protein G agarose preblocked with 5% BSA/PBS and salmon sperm DNA. Protein Co-IPs were processed by standard methods and elutions were analyzed by western blot. For RNA immunoprecipitations (RIPs), beads were treated with proteinase K for 10 min, and RNA was eluted using the Trizol method and nucleic acids were precipitated in the presence of glycogen. RT-PCR was performed using the oligos described above.
8
Sam68 Protein Interactome Profiling
9
Aβ Quantification in BMEC Secretome
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Cell conditioned medium from BMECs monolayers grown at Day 10 were collected and immediately frozen at − 80 ºC. Aβ1–40 and 1–42 levels in supernatants were measured using their respective ELISA Quantikine(R) kits (R&D Systems, Minneapolis, MN).
In experiments involving cell homogenates, cells were briefly washed with PBS followed by homogenization using 200 µL RIPA buffer (ThermoFisher) supplemented with 1× protease inhibitors cocktail (ThermoFisher). Cells were maintained on ice for 10 min, followed by centrifugation at 14,000 rpms for 10 min at 4 ºC. The supernatant of such lysates was collected and immediately frozen at − 80 ºC pending analysis. Cell lysates protein concentrations were measured using BCA assay and normalized (using the diluent provided by the kit) to achieve a final protein concentration of 1 µg total protein/µL if necessary. 200 µL of such normalized samples were loaded onto the ELISA plate.
In experiments involving cell homogenates, cells were briefly washed with PBS followed by homogenization using 200 µL RIPA buffer (ThermoFisher) supplemented with 1× protease inhibitors cocktail (ThermoFisher). Cells were maintained on ice for 10 min, followed by centrifugation at 14,000 rpms for 10 min at 4 ºC. The supernatant of such lysates was collected and immediately frozen at − 80 ºC pending analysis. Cell lysates protein concentrations were measured using BCA assay and normalized (using the diluent provided by the kit) to achieve a final protein concentration of 1 µg total protein/µL if necessary. 200 µL of such normalized samples were loaded onto the ELISA plate.
10
Protein Extraction from BIAHR and H
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The protein extraction was performed as previously described7 (link). Briefly, BIAHR and H proteins were solubilized with a commercially available T-PER lysis buffer (Thermo Fisher Scientific, France) complemented with a protease inhibitors cocktail (Thermo Fischer scientific, France). Solubilization was performed using a Polytron TP-20 Homogenizer 8 (Kinematica, Lucerne, Switzerland). After two centrifugations, at 13,000 g for 45 min at 4 °C to discard insoluble material from the supernatant, the protein concentration of every soluble extract was determined using a Bradford assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific, France).
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