5 × 106 cells were lysed in 400 µl of Tris lysis buffer (40 mM Hepes, 1 mM EDTA, 120 mM NaCl, 10 mM tetrasodium pyrophosphate, 10% glycerol, 1% Triton X-100, and 0.1% SDS) supplemented with antiphosphatases (Phosphatase Inhibitor Cocktail, MilliporeSigma; 1 mM Na3VO4, 5 mM β-glycerophosphate, 1 mM PMSF) and antiprotease cocktail (DA36046; Roche). Cell lysates were immunoprecipitated with antibody against Slik for 4 h and then incubated with protein A sepharose beads for 2 h. The beads from immunoprecipitations were subjected to an in vitro kinase assay as previously described (Plutoni et al., 2019 (link)).
Anti protease cocktail
Manufactured by Roche
Sourced in United States, France, Switzerland, Germany
The Anti-protease cocktail is a laboratory reagent designed to inhibit the activity of various proteases, which are enzymes that break down proteins. This product can be used to protect protein samples from degradation during extraction, purification, or other analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (3 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Superdex 200 column, by GE Healthcare (2 mentions)
Omni ceramic homogenization beads, by Omni International (2 mentions)
Omni bead ruptor, by Roche (2 mentions)
Pierce bca protein quantitation kit, by Thermo Fisher Scientific (2 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ecl chemiluminescence detection system, by PerkinElmer (2 mentions)
Hybond c extra nitrocellulose membrane, by GE Healthcare (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Pen strep and trypsin edta, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Ampliqon (2 mentions)
Polyvinylidene uoride pvdf, by Roche (2 mentions)
Bovine serum albumin phenyl methyl sulfonyl uoride pmsf, by Roche (2 mentions)
P ampk, by Cell Signaling Technology (2 mentions)
P acc, by Cell Signaling Technology (2 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ecl reagent, by GE Healthcare (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modi ed eagle s medium, by Thermo Fisher Scientific (2 mentions)
48 protocols using anti protease cocktail
1
In Vitro Kinase Assay for Slik
2
Isolation and Characterization of Viral Protein Aggregates
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Treatment were performed as described in [66 (link)]. Briefly, 2x10^7 RAW264.7 cells were plated onto 10cm dishes, Mock or MNV-infected the next day at a MOI of 10 and further cultivated for 10h post infection (p.i). The dishes were placed on ice and washed with cold PBS (from 10x solution–Lonza) and the cells lysed on ice with 1ml of EE buffer (Hepes pH 7.4 50mM, NaCl 200mM, Igepal 0.1%, EDTA pH8 1mM, EGTA pH8 2.5mM, Glycerol 10%, DTT 1μM supplemented with RNAsin (Promega) and anti-protease cocktail (Roche)). Lysates were transferred to a 1.5ml Eppendorf and incubated with agitation for 20min at 4°C before being spun down at 13,000 rpm 15min at 4°C. 50μl of the supernatant was kept as “input”, mixed with an equal volume of 2x Loading buffer (Cell Signaling) and boiled at 95°C for 5min. The remaining supernatant was supplemented with 100μM of b-isox (Sigma) and the precipitation reactions were carried out for 90min at 4°C with agitation. Aggregates were pelleted down by centrifugation at 10,000xg for 10min at 4°C. 50μL of the supernatant was kept as “soluble fraction”, mixed with an equal volume of 2x loading buffer and boiled at 95°C for 5min. The pellets were washed twice with 500μL of cold EE buffer, spin down at 10,000g 10min at 4°C, resuspended into 200μL of 1x Gel Loading Buffer (New England Biolabs) as “insoluble fraction” and boiled at 95°C for 5min.
3
Yeast Cell Lysis and Polysome Profiling
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A total of 18 OD600 of exponentially growing yeast cells were treated for 10 min with 10 μg/ml of cycloheximide, harvested by centrifugation, and washed cell pellets were resuspended in 150 μl lysis buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 30 mM MgCl2, 200 μg/mL heparin, 10 μg/mL cycloheximide, 1 mM DTT, 40 U/mL RNase inhibitor, antiprotease cocktail (Roche), in DEPC-treated water). After the addition of 425–600 μm glass beads (Sigma Aldrich), cells were lysed by the alternating of 6 vortexing and ice-cooling steps of 30 sec each and then centrifuged for 5 min at 2,300 g at 4 °C. Supernatants were recovered, centrifuged at 9,300 g for 10 min at 4 °C and assayed for RNA content at OD260. 100 U OD260 of RNA were loaded on top of 10–40% sucrose gradients, centrifuged for 3h at 41,600 rpm using a SW55ti swing rotor (Beckman coulter). 200 μL fractions were collected, and OD260 was measured on 1/10 diluted fractions26 (link)72 (link).
4
Western Blot Analysis of Protein Expression
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Cells were lysed in a 1% SDS (v/v) extraction buffer containing an anti-protease cocktail (Roche). Protein concentrations were determined using the Bradford assay (MicroBCA, Pierce). After resolution by SDS-PAGE, electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes that were blocked and probed with the following antibodies: GRP94 (1:200, sc1794, Santa Cruz Biotechnology), HexA (1:100, sc-376777, Santa Cruz Biotechnology), GM2-AP (1:100, sc-514437, Santa Cruz Biotechnology), GAPDH (1:2500, BD Pharmingen) and the corresponding peroxidase-conjugated secondary antibodies at 1:2000. Immunoreactive bands were quantified using a VersaDoc™ (Bio-Rad) Imaging System using the Super Signal west-Pico (Pierce). Molecular weights were established with See Blue Plus2 Pre-stained Standard (Invitrogen).
5
Quantification of BIM Protein Expression in KG-1 Cells
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The BIM protein expression was determined by Western blotting analysis. KG-1 cells were seeded in a 6-well plate at a density of 5×105 cells/well and incubated for 24 h. To evaluate the amount of BIM protein in KG-1 cells, total cell protein was extracted by NP40 lysis buffer after 24h treatment. To prevent protein degradation, phosphatase inhibitors (Sigma, USA) and anti-protease cocktail (Roche, Germany) were used. The concentration of extracted protein was assessed by Qubit Protein Assay Kit (Invitrogen, USA). A total of 40 µg protein/sample was used for gel electrophoresis (on 7.5 % and 12 % SDS-PAGE gel) and the resultant proteins were transferred to nitrocellulose membrane. Blocking was performed in 5 % BSA blocking buffer. Then, the blots were incubated with primary antibody at a dilution of 1:1000 at 4 °C overnight and again incubated with secondary antibody at a dilution of 1:2000. Protein detection was carried out ECL Western Blot Detection Reagents.
6
Purification and characterization of STARD4 and mutants
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STARD4 and STARD4
mutants (K49A/K52A, K219A, M206C, L124C, and L124D)
in the pET-SUMO vector were expressed in Escherichia coli BL21(DE3) cells during overnight incubation at 18 °C. Bacterial
pellets were resuspended in 20 mM HEPES (pH 7.2), 100 mM KCl, 20 mM
imidazole, 1 mM TCEP, and 0.1% IGEPAL supplemented with an anti-protease
cocktail (Roche) and 1 mM phenylmethanesulfonyl fluoride. The resuspended
cell pellets were lysed on ice by sonication followed by ultracentrifugation
at 100000g for 1 h. The supernatant was incubated
with pre-equilibrated Ni-NTA resin under constant agitation at 4 °C
for 1 h. Following incubation, the supernatant/resin slurry was passed
through a column, and the column was washed with 20 mM HEPES (pH 7.2),
100 mM KCl, 20 mM imidazole, and 1 mM tris(2-carboxyethyl)phosphine
(TCEP). The SUMO–STARD4 protein was eluted with 20 mM HEPES
(pH 7.2), 100 mM KCl, 0.5 M imidazole, and 1 mM TCEP. The eluted protein
was dialyzed overnight at 4 °C in 20 mM HEPES (pH 7.2), 100 mM
KCl, and 1 mM DTT in the presence of the Ulp1 protease to remove the
His-SUMO tag. STARD4 was further purified using size exclusion chromatography
using a Superdex200 column (GE Healthcare) in 20 mM HEPES (pH 7.2),
100 mM KCl, and 1 mM DTT. Purified protein was stored at −80
°C.
mutants (K49A/K52A, K219A, M206C, L124C, and L124D)
in the pET-SUMO vector were expressed in Escherichia coli BL21(DE3) cells during overnight incubation at 18 °C. Bacterial
pellets were resuspended in 20 mM HEPES (pH 7.2), 100 mM KCl, 20 mM
imidazole, 1 mM TCEP, and 0.1% IGEPAL supplemented with an anti-protease
cocktail (Roche) and 1 mM phenylmethanesulfonyl fluoride. The resuspended
cell pellets were lysed on ice by sonication followed by ultracentrifugation
at 100000g for 1 h. The supernatant was incubated
with pre-equilibrated Ni-NTA resin under constant agitation at 4 °C
for 1 h. Following incubation, the supernatant/resin slurry was passed
through a column, and the column was washed with 20 mM HEPES (pH 7.2),
100 mM KCl, 20 mM imidazole, and 1 mM tris(2-carboxyethyl)phosphine
(TCEP). The SUMO–STARD4 protein was eluted with 20 mM HEPES
(pH 7.2), 100 mM KCl, 0.5 M imidazole, and 1 mM TCEP. The eluted protein
was dialyzed overnight at 4 °C in 20 mM HEPES (pH 7.2), 100 mM
KCl, and 1 mM DTT in the presence of the Ulp1 protease to remove the
His-SUMO tag. STARD4 was further purified using size exclusion chromatography
using a Superdex200 column (GE Healthcare) in 20 mM HEPES (pH 7.2),
100 mM KCl, and 1 mM DTT. Purified protein was stored at −80
°C.
7
Comprehensive Cell Lysis and Nuclear Extraction
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Total cell lysis was performed with a RIPA buffer composed of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM sodium orthovanadate, in the presence of an antiprotease cocktail (Roche Diagnostics, Mannheim, Germany). Nuclear lysates were performed as described [74 (link)] with slight modifications. Briefly, keratinocytes were lysed in an hypotonic buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, and 0.1 mM EGTA) supplemented with 1 mM sodium orthovanadate and an anti-protease cocktail. The lysate was centrifuged at 1,800xg for 10 min to precipitate nuclei. The nuclear pellet was finally lysed by an hypertonic buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, in the presence of 1 mM sodium orthovanadate and an antiprotease cocktail). During all the phases, cell lysates were kept on an ice bath.
8
Cell Homogenization and Protein Extraction
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The cell homogenization was performed as previously described [56 (link)]. Briefly, 5x106 cells were homogenized in lysis buffer (8M urea/2M thiourea (GE Healthcare, Uppsala, Sweden), 4% CHAPS (Sigma Aldrich, Saint Louis, MO, USA), 50 mM DTT, anti-protease cocktail (Roche Diagnostics, Mannheim, Germany)) before protein precipitation using the 2D clean-up kit (GE Healthcare). The supernatant was removed and the pellet was suspended in 250 μl of sample buffer (8M urea/2M thiourea, 4% CHAPS). The protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).
9
Protein Expression Analysis via SDS-PAGE and Western Blot
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To assess protein expression in the different experiments, cell lines were lysed in a 1% SDS (v/v) extraction buffer containing an anti-protease cocktail (Roche, Vilvoorde, Belgium). Protein concentrations were determined using the Bradford assay (MicroBCA, Pierce, Belgium). After resolution by SDS-PAGE, electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes that were blocked and probed with the following antibodies: ACOT7 (1:1000, Abcam, Cambridge, UK), LXRα (1:1000, PPMX, Tokyo, Japan), SREBP-1 (1/500, Abcam), GRP94 (1/1000, Sta Cruz, CA, USA), Mfn1 (1:1000, Santa Cruz, CA, USA,), Mfn2 (1:2000, Abcam), VDAC (1:5000, Abcam), α-tubulin (1:10,000, Sigma), actin (1:2000, Sigma) and the corresponding peroxidase-conjugated secondary antibody at 1:2000: Peroxidase conjugated anti-rabbit secondary Ab (Amersham, Little Chalfont, UK), Peroxidase conjugated Antimouse secondary Ab (Pierce, Perbio Science Ltd., Cheshire, UK). Immunoreactive bands were quantified using a VersaDoc™ (BioRad Laboratories, Hercules, CA, USA) Imaging System using the Super Signal west-Pico (Pierce Biotechnology, Rockford, IL, USA). MWs were established with See Blue Plus2 prestained Standford (Invitrogen).
10
TRAF6 Immunoprecipitation Protocol
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The SH-SY5Y cells were cotransfected with empty vector Flag or Flag-TRAF6 through FuGENE HD (Promega), following the manufacturer's instructions. The cells were lysed in lysis buffer (20 mM Na-Hepes, pH 7.7; 225 mM KCl, 1% Triton X-100) supplemented with an anti-protease cocktail (Roche, Basel, Switzerland). The cell lysates were incubated with an ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) for 16 h at 4 °C. After washing, the immunoprecipitated proteins were eluted with 2x sodium dodecyl sulfate (SDS) sample buffer, boiled at 95 °C, and analyzed by Western blot.
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