The BioID expression vector ligated with α-syn WT or A53T was transfected into SH-SY5Y cells. Biotinylation was induced by treatment with 50 μM biotin. Cells were washed with ice-cold PBS and harvested in the lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.4% (w/v) SDS, and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA)) 24 h after biotinylation. After sonication, the lysates were then centrifuged at 22,250× g for 20 min. Biotinylated proteins in the supernatants were captured on streptavidin beads by rotation overnight at 4 °C. The following day, the beads were pelleted and washed thrice with wash buffer A (50 mM HEPES, 500 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA)) and twice with wash buffer B (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA)). Beads were then mixed with 2× SDS sample buffer (Bio-Rad, Hercules, CA, USA) and boiled at 95 °C for 15 min. The reaction was verified via streptavidin immunoblot analysis; coomassie-stained gel was used, in combination with mass spectrometry, to identify the proteins.
Protease inhibitor cocktail
Manufactured by GenDEPOT
Sourced in United States
Protease inhibitor cocktail is a laboratory reagent used to prevent the degradation of proteins by proteolytic enzymes. It contains a mixture of chemical compounds that inhibit the activity of various proteases. The core function of this product is to maintain the integrity of protein samples during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Phosphatase inhibitor cocktail, by GenDEPOT (8 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Nitrocellulose membrane, by GE Healthcare (5 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Bca assay kit, by Thermo Fisher Scientific (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Westsave, by AbFrontier (3 mentions)
Ripa buffer, by GenDEPOT (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Protein a g agarose, by Thermo Fisher Scientific (2 mentions)
Monoclonal mouse anti ha agarose beads, by Merck Group (2 mentions)
Monoclonal mouse anti myc antibodies, by Takara Bio (2 mentions)
86 protocols using protease inhibitor cocktail
1
Proteomic Analysis of α-Synuclein Interactome
2
Subcellular Fractionation of α-Synuclein Variants
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SH-SY5Y cells transfected with either GFP-tagged α-syn WT or A53T were subjected to subcellular fractionation. Harvested cells were mixed with buffer A (0.01 M HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.075% (v/v) NP40), containing a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA), and incubated for 10 min on a rotator. After centrifugation at 750× g for 10 min, the supernatants containing cytoplasmic proteins were transferred to new tubes placed on ice. The remaining pellets were suspended in buffer B (0.01 M HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.075% (v/v) NP40, and 0.25 M sucrose), containing a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA), and incubated on a rotator for 20 min, followed by centrifugation at 750× g for 10 min. After removing the supernatants, the pellets were suspended in buffer A to dissolve the nuclear proteins.
3
Immunoprecipitation of Giardia Proteins
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Giardia cells carrying pGlPLK-HA.neo and pGlKin-13-Myc.pac were harvested, washed 3 times with ice-cold PBS, and lysed with lysis buffer (10 mM Tris-Cl, 5 mM EDTA, 130 mM NaCl, and 1% Triton X-100, pH 7.4) containing protease inhibitor cocktail (GenDEPOT, Katy, Taxas, USA). Lysates precleared using Protein A/G Agarose (Thermo Fisher Scientific) for 1 h at 4°C were incubated with monoclonal mouse anti-HA agarose beads (Sigma-Aldrich) or monoclonal mouse anti-Myc antibodies (Clontech, Mountain View, California, USA) at 4°C overnight. Beads were washed with immunoprecipitation washing buffer (50 mM Tris-Cl, 150 mM NaCl, and 1% Triton X-100, pH 7.4) and resuspended in 2×SDS sample buffer. The samples were analyzed by Western blotting using anti-HA or anti-Myc antibodies.
4
Cell Lysis and Western Blot Analysis
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Cells were lysed on ice for 30 min in a buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5% NP-40, 0.1 mM Na3VO4, 50 mM NaF, 30 mM Na4O7P2 · 10 H2O, and a protease inhibitor cocktail (GenDepot, Barker, TX, USA). Equal amounts of protein in the lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and analyzed using specified antibodies with an ECL detection system (GE Healthcare, Chicago, IL, USA).
5
Oxidative Stress Modulation in C2C12 Cells
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C2C12 cells were pretreated with either 3–30 μg/mL of CM-SD or 30 μM of IsoR for 1 h, and subsequently exposed to 100 μM of H2O2 for 12 h. After treatment, the cells were lysed by a radioimmunoprecipitation assay buffer containing 1 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) for 1 h on ice and centrifuged at 15,000× g for 10 min. MDA levels in the resulting lysates (approximately 250 μg of protein lysates) were assayed according to the manufacturer’s instruction (TBARS Assay Kit; Cayman Chemical, Ann Arbor, MI, USA). The absorbance of the sample was monitored at 530 nm using a microplate reader (BioTek Instruments, Inc.), and the concentration of MDA was determined from a standard curve.
6
Evaluating Cell Signaling Pathways in Compound-Treated DU145 Cells
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Compound-treated DU145 cells were lysed with RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1% protease inhibitor cocktail (GenDEPOT, Katy, TX, USA). The lysed cells were incubated on ice for 20 min and centrifuged at 4 °C for 20 min at 12,000 rpm to obtain the supernatant. The protein was quantitated using a PierceTM BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Samples were mixed with 2× loading buffer (1 mM Tris-HCl, pH 6.8, glycerol, 10% SDS, bromophenol blue, beta-mercaptoethanol) and heated at 98 °C for 3 min. Samples were separated by SDS-PAGE and transferred to a PVDF membrane, and the membrane was incubated overnight with primary antibodies at 4 °C. Anti-topoisomerase IIα (#12286), anti-Cyclin D1 (#2922), anti-cleaved-PARP (#9541), anti-Bax (#2772), anti-Bcl-2 (#2872), anti-β-actin (#4967) and α-Tubulin (#2144) were from Cell Signaling Technology (Danvers, MA, USA). Anti-p27kip1 (#E11-0965B) was from EnoGene Biotech Co, Ltd. (NY, USA). The images were scanned by LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan) and analyzed using Multi-Gauge Software (Fuji Photo Film Co., Ltd., Tokyo, Japan).
7
Immunoprecipitation of PKA Complexes
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B16F0 cells were disrupted using a lysis buffer (50 mM Hepes pH 7.5, 0.1% NP-40, 5 mM EDTA, 150 mM NaCl, and 1 mM DTT) supplemented with protease inhibitor cocktail (GenDepot, P3100). Whole cell extracts were incubated with anti-PKA-RIIβ antibody (Santa Cruz Biotechnology, sc-376778) overnight at 4°C and then with protein G-sepharose beads (GE healthcare, 17-0618-01) at 4°C for another 2 h. These IP complexes were resolved on SDS-polyacrylamide gels by electrophoresis and probed with another antibody against anti-PKA-Cα (Cell Signaling Technology, 4782) to detect the co-precipitates.
8
Protein Expression Analysis in Lung Fibroblasts
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The right inferior lobe lung and MRC5 fibroblasts were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (100 μL/mL, GenDEPOT, Katy, TX, USA). Equal quantities of protein were separated on 6–10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 2 h at 37°C and then incubated with primary antibodies (α-SMA, TGF-β receptor I (TGFβRI), p-Smad2, Smad2, p-Smad3, Smad3, tissue inhibitor of metalloproteinase 1 (TIMP-1), and matrix metalloproteinase 9 (MMP-9), collagen I, and GAPDH; Abcam) overnight at 4°C. After exposure to HRP-conjugated secondary antibody for 2 h at 37°C, the membranes were analyzed with ECL reagents (Millipore). Relative protein expression levels were determined by scanning densitometry (ChemiDoc XRS + Systems, Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using Image Lab 5.0 software (Bio-Rad Laboratories).
9
Protein Extraction and Western Blot
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Cells were lysed in RIPA buffer (150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5 and 2 mM EDTA) (GenDEPOT, TX, USA) containing 1% protease inhibitor cocktail (GenDEPOT). 20 μg of each sample is separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). Western blotting was performed as previously described by Liu et al.55 (link).
10
TA3 Regulation of MG63 Cell Signaling
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MG63 cells were seeded in 6-well dishes (1.5 × 105). Then, the cells were treated with TA3 (0, 5, and 10 μM) for 24 h. Then, MG63 cells were collected and lysed in RIPA buffer (with phosphatase inhibitor cocktail) (Gendepot, USA) and protease inhibitor cocktail (Gendepot, USA). The lysates were normalized by bicinchroninic acid (BCA) protein assay kit (Thermo Fisher, USA). The samples were resuspended in SDS-sample buffer [Tris-HCl (62 mM), pH 6.8, ethylenediaminetetraacetic acid (EDTA) (1 mM), glycerol (10 %), SDS (5%), dithiothreitol (50 mM)]. Then, SDS-PAGE was performed. The protein bands of the gel were transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membranes were incubated in blocking buffer (5% nonfat skim milk in 1X tris-buffered saline (TBS)-T) for 1 h at 4°C on shaking incubator and incubated with specific primary antibodies at 4°C overnight. Then, the membranes were subjected to the incubation with secondary antibodies for 2 hrs. The membranes were washed with TBS-T three times. The immunoreaction was measured using Western blot detection kit (AbFrontier).
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