Hearts were harvested and flash-frozen. TES buffer (20-mmol/l Tris, 250-mmol/l sucrose, 1-mmol/l EDTA, pH 7.4) with 1× complete protease (4693132001, Sigma-Aldrich) and phosphatase inhibitor tablets (4906837001, Sigma-Aldrich) was used for protein homogenization in a bead beater tissue homogenizer (BioSpec). Homogenized tissue samples were heated to 70°C in 2× Laemmli and ran on a 4% to 15% TGX gel (Bio-Rad).
Complete protease
Manufactured by Merck Group
Complete protease is a laboratory reagent used for the digestion and processing of protein samples. It is a mixture of enzymes that cleave peptide bonds, breaking down proteins into smaller fragments. The core function of Complete protease is to facilitate the preparation and analysis of protein samples for various applications in biochemistry and molecular biology research.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop phosphatase inhibitor, by Merck Group (3 mentions)
High select fe nta phosphopeptide enrichment kit, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Stagetip empore c18 material, by 3M (2 mentions)
Lys c protease, by Fujifilm (2 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Edta free, by Merck Group (2 mentions)
Restore plus buffer, by Thermo Fisher Scientific (2 mentions)
Phosstop, by Merck Group (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Bead beater tissue homogenizer, by Biospec (1 mentions)
Tgx gel, by Bio-Rad (1 mentions)
Gfp trap beads, by Proteintech (1 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (1 mentions)
Gs710 densitometer, by Bio-Rad (1 mentions)
Pierce coomassie plus, by Thermo Fisher Scientific (1 mentions)
Phosphatase, by Merck Group (1 mentions)
Western lightning plus ecl reagent, by PerkinElmer (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
12 protocols using complete protease
1
Cardiac Tissue Homogenization and Protein Extraction
2
CREB-HD phosphorylation dynamics analysis
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Cells transfected with expression vectors for wt and mutant CREB-HΔTMC were washed with ice-cold PBS, scraped, and pelleted. After suspension in PBS, samples were lysed in buffer for λ phosphatase containing 0.4% NP-40, 0.5% Triton X-100, protease inhibitors (1× Complete Protease; Sigma-Aldrich), and 1 mM phenylmethylsulfonyl fluoride. As controls, two aliquots of the sample were treated with orthovanadate (10 mM) and sodium fluoride (20 mM) and incubated at 37°C either with or without added phosphatase for 30 min to reflect the starting material before incubation with phosphatase alone. Parallel samples were then incubated with 400 U of λ phosphatase and incubated at 37°C for different times up to 30 min. The reactions were terminated by adding SDS sample buffer to 1× and boiling for 5 min. Samples were analyzed by SDS–PAGE and Western blotting.
3
Affinity Purification of EGFP-PLK3
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HEK293 cells stably expressing EGFP or EGFP-PLK3 were extracted by IP buffer (20 mM HEPES pH 7.5, 10% glycerol, 150 mM NaCl, 0.5% NP40) supplemented with cOmplete protease and PhosSTOP phosphatase inhibitors (Sigma) and sonicated for 3 × 20 s on ice. Cell extracts were cleared by centrifugation at 15,000 rpm 10 min at 4 °C and incubated with GFP-Trap beads (Chromotek, Planegg, Germany) for 2 h. After three washes in IP buffer, bound proteins were eluted from beads by Laemli buffer and analyzed by immunoblotting. Alternatively, bound proteins were analyzed by mass spectrometry using Orbitrap Fusion (Thermo Scientific). Proteins bound to EGFP-PLK3 that were enriched compared to the empty EGFP control in at least two out of three independent experiments were considered as potential interactors and were validated by immunoprecipitation followed by immunoblotting. For in vitro kinase assay, wild-type or mutant EGFP-PLK3 was immunoprecipitated using GFP Trap, washed three times in IP buffer and incubated with casein in kinase buffer (10 mM HEPES pH 7.4, 5 mM MgCl2, 2 mM EGTA, 1 mM DTT, 2.5 mM β-glycerolphosphate, 100 µM ATP and 5 µ Ci 32P-γ-ATP) for 20 min at 30 °C. Proteins were separated using SDS-PAGE, and phosphorylation was visualized by autoradiography.
4
Western Blotting Analysis of PMP22 in Sciatic Nerve
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For Western blotting, mouse sciatic nerve lysates from wild-type, C22, or PMP22-knockout mice (4 μg/lane) were blotted with anti-human PMP22-specific polyclonal antibody.25 Confluent cultures of dermal fibroblasts, after 48 hours incubation in 2% FCS containing medium, were lyzed in sample buffer [62.5 mmol/L Tris, 3% SDS, and 10% glycerol, with complete protease (Roche) and phosphatase (Sigma-Aldrich, St. Louis, MO) inhibitors]. Protein concentration was determined using the Bradford method (Pierce Coomassie Plus; Thermo Fisher Scientific, Waltham, MA), and samples were denatured by adding β-mercaptoethanol and boiling for 10 minutes. Equal protein amounts were resolved on SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were rinsed in Tris-buffered saline with 0.05% Tween-20, blocked in 5% nonfat milk, and then probed with the indicated primary antibodies (Table 2 ) overnight at 4°C. Bound antibodies were detected using Western Lightning Plus ECL reagents (Perkin Elmer, Waltham, MA). Films were digitally imaged using a GS-710 densitometer (Bio-Rad Laboratories, Hercules, CA) or a ChemiDoc MP Imaging System (Bio-Rad Laboratories) and analyzed using ImageJ software version 1.45 (NIH, Bethesda, MD; http://imagej.nih.gov/ij ). All biochemical experiments were repeated at least three times using protein lysates from independent cultures.
5
Hippocampal Synaptosome Isolation
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Synaptosome extracts were prepared as described previously [59 ]. Briefly, the hippocampus was homogenized (using a Dounce homogenizer) in DEPC-treated water (Ambion) supplemented with 0.32 M sucrose, 20 mM Tris–HCl, 0.5 M EDTA, and 0.5 M EGTA (pH 7.4), containing complete protease (Sigma) and phosphatase inhibitor cocktails (Sigma). After homogenization, the crude synaptosomal fraction (synaptosomes plus mitochondria) was isolated by two sequential centrifugations (1,500 × g, 10 min followed by 12,500 × g, 20 min; at 4 °C). The protein content of the synaptosomal fractions was determined using the Bradford assay. For western blot experiments, synaptosomal preparations were temporally stored at −80 °C.
6
Immunoblotting Analysis of Cell Signaling
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Cells were washed twice with a PBS- 0.5% EGTA solution and lysed in RIPA buffer (150 mM NaCl, 1% NP40, 100 mM Tris pH 7.5, 0.1% SDS, 0.5% DOC, 1 mM EGTA) supplemented with complete protease (Roche) and phosphatase inhibitors (Sigma). After clarification by centrifugation at 14 000 rpm for 20 min at 4°C, cell extracts were subjected to SDS-PAGE. Proteins were revealed with mouse monoclonal anti-E-cadherin (clone 36, Becton Dickinson), anti-β-catenin (clone 14, Becton Dickinson), anti-fibronectin (clone 10, Becton Dickinson), anti-vimentin (clone V9, Dako), anti-N-cadherin (Becton Dickinson), anti-phospho-ERK1/2 (9106S, Cell Signaling), anti-GAPDH (6C5, Biodesign), rabbit monoclonal anti-active caspase 3 (ab32042, Abcam), or rabbit polyclonal anti-SNAIL1 (ab17732, Abcam), anti-SNAIL2 (G-18/SC-10436, Tebu-bio), anti-SNAIL3 (HPA016757, Sigma), anti-ERK1/2 (#9102, Cell Signaling), anti-AKT (8272, Cell Signaling), anti-phospho-AKT (Ser473) (#4058, Cell Signaling), anti-c-MYC A14 (sc-789, Santa Cruz Biotechnology), anti-HA Y11 (Santa-Cruz) and horseradish-peroxidase-conjugated secondary antibodies (Dako). Antigen-antibody complexes were revealed with a reagent for western blotting (Santa Cruz).
7
Phosphoproteome Analysis of Yeast Samples
8
Phosphoproteomics Sample Preparation
9
Immunostaining of Electroporated Retinal Tissue
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The immunostaining protocol was adapted from Robichaux, et al. (41 (link)). Electroporated retinas with mCherry expression were dissected in supplemented mouse ringers, pH 7.4 and approximately 313–320 mOsM. Fresh retinas were blocked in 10% normal donkey serum, 0.3% saponin, 1 × cOmplete Protease (Millipore Sigma) diluted in supplemented mouse ringers for 2 hours at 4°C. Primary antibodies diluted in block were incubated for 20–22 hours, rinsed and incubated in secondary antibodies for 2 hours, all at 4°C. Retinas were rinsed, fixed in 4% paraformaldehyde for 30 minutes at 22°C, embedded in 4% agarose (Thermo Fisher Scientific), after which 100 μm vibratome retinal-sections were collected.
10
Protein Extraction and Immunoblot Analysis
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Cells were lysed on ice for 10 minutes in freshly prepared Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with 1X Protease Complete, EDTA-free (Sigma) and 1X PhosSTOP (Sigma) before centrifugation at 15,000 rpm for 12-min at 4°C. Protein lysates were prepared using 4× Laemmli sample buffer (BioRad) heated in boiling water for 5-min. Immunoblot analysis was conducted with the antibodies listed in Supplementary Table S5 . Immunoblot analysis of Akt phosphorylation was performed sequentially using the same PVDF membrane, first probing for phosphorylated Akt (Ser473), then total Akt. PVDF membranes were stripped for 8-min using Restore PLUS buffer (Thermo Scientific) and blocked with 5% BSA between subsequent probes. Tubulin was used a loading control for all immunoblot experiments. Images were digitally captured using a Bio-Rad ChemiDoc MP and analyzed with ImageJ.
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