Extracted proteins were precipitated using a Calbiochem ProteoExtract® Protein Precipitation Kit as per manufacturer’s instructions (cat# 539180, Merck, Auckland, NZ). The protein pellet was dissolved in a digestion buffer of 8 M urea and 100 mM Tris–HCl, pH 8.5. After a second Bradford assay, 20 µg of protein was taken from each sample and the volume brought to 25 µL using digestion buffer. Subsequently, the proteins were reduced, alkylated and digested as previously described [29 (link), 30 (link)]. The resulting tryptic peptides were purified using OMIX C18 100 µL zip-tips (cat# A57003100K, Agilent Technologies, Santa Clara, CA, USA). Eluates were pooled and dried down using a vacuum centrifuge, then brought to 100 µL using 0.1% formic acid (FA) in 3% acetonitrile (ACN).
Proteoextract protein precipitation kit
Manufactured by Merck Group
Sourced in United States, New Zealand
The ProteoExtract Protein Precipitation Kit is a laboratory product designed to effectively precipitate proteins from various biological samples. The kit utilizes a proprietary precipitation reagent to selectively precipitate proteins, enabling their efficient separation and concentration for further analysis.
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Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Trypsin lys c, by Promega (1 mentions)
Rnaseout ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Gfp trap, by Proteintech (1 mentions)
Mammalian protease inhibitor cocktail, by Merck Group (1 mentions)
Speedvac, by Labconco (1 mentions)
Trypsin, by Promega (1 mentions)
Protein lobind tube, by Eppendorf (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Oasis hlb 30 mg sorbent cartridges, by Waters Corporation (1 mentions)
Ecl plus system, by GE Healthcare (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
8 protocols using proteoextract protein precipitation kit
1
Protein Extraction, Reduction, and Digestion Protocol
2
Protein Extraction and Digestion
3
Proteomic profiling of MUT-16 interactome in C. elegans
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~500,000 synchronized N2 (wild-type) or USC717 (mut-16(cmp3[mut-16::gfp::3xFLAG + loxP])) adult C. elegans (~68 hr at 20°C after L1 arrest) were collected in IP Buffer (50 mM Tris-Cl pH 7.4, 100 mM KCl, 2.5 mM MgCl2, 0.1% Igapal CA-630, 0.5 mM PMSF (0.5 mM), cOmplete Protease Inhibitor Cocktail (Roche 04693159001), and RNaseOUT Ribonuclease Inhibitor (ThermoFisher 10777019)), frozen in liquid nitrogen, and homogenized using a mortar and pestle. After further dilution into IP buffer (1:10 packed worms:buffer), insoluble particulate was removed by centrifugation and a sample was taken as ‘input.’ The remaining lysate was used for the immunoprecipitation. GFP and FLAG immunoprecipitation was performed at 4°C for 2 hr using anti-GFP affinity matrix [RQ2 clone] (MBL International D153-8) and anti-FLAG affinity matrix [M2 clone] (Sigma-Aldrich A2220), then washed 10 times in immunoprecipitation buffer. After immunoprecipitation, samples were precipitated using the ProteoExtract Protein Precipitation Kit (EMD Millipore 539180) and submitted to the Taplin Mass Spectrometry facility at Harvard Medical School for protein identification.
4
Affinity Purification of GFP-Interacting Proteins
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Preparation and freezing of synchronized worms were carried out as in Cheeseman et al., (2004) (link) except that worms were grown on 8P plates seeded with the E. coli strain NA22 at room temperature. Worm lysis was carried out by grinding worm pellets in liquid nitrogen and subsequent sonication (Diagenode) was performed with the following settings: 3× 5 cycles on high with 30 seconds on and 30 seconds off. Lysate was centrifuged for 45 minutes at 15000 rpm at 4°C. To avoid unspecific protein binding the worm lysate was pre-cleared by incubation to uncoupled agarose beads (Chromotek, bab-20) for 1 hour at 4°C. Pre-cleared worm lysate was incubated with GFP-trap (Chromotek, gta-20) for 3 hours at 4°C. Beads were washed 3 times with lysis buffer containing proteinase inhibitors (Roche) and 7 times with lysis buffer without proteinase inhibitors. Proteins were eluted by adding 40 μl 2xLSB and boiling for 5 min at 95°C. Protein precipitation was carried out using ProteoExtract Protein Precipitation Kit (EMD Millipore, 539180–1KIT) according to the manufacturer’s protocol and submitted for MS analysis to the Taplin Biological Mass Spectrometry Facility.
5
Protein Precipitation and Quantification
6
Proteome Extraction and Tryptic Digestion
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Using ProteoExtract Protein Precipitation Kit (Merck Ltd., Auckland, New Zealand) 30 µg of proteins were precipitated according to the manufacturer’s instructions and resuspended in 100 mM Tris-HCl, pH 8.8, and 8 M urea and then digested with trypsin (v5111, Promega, Madison, WI, USA). Purification of resulting tryptic peptides was accomplished with OMIX C18 Ziptips (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer’s protocol. Peptides were eluted in 20 μL of 0.15% formic acid (FA) in 50% acetonitrile (ACN) and 20 μL of 0.15% FA in 70% ACN, respectively, which were subsequently combined and dried down to ~10 μL at 35 °C using SpeedVac (Labconco, Kansas, MO, USA) in a 0.5 mL Protein LoBind® tube (Eppendorf, Hamburg, Germany). The peptides were placed in 0.1% FA in water to make a final solution volume of 140 μL, vortexed and centrifuged at room temperature and then transferred to a glass vial with a glass inserts (ThermoFisher Scientific, Auckland, New Zealand) for liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis in triplicate.
7
Protein Extraction and Tryptic Digestion
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Protein samples were precipitated with a ProteoExtract Protein Precipitation Kit (539180) from MilliporeSigma as per the manufacturer’s protocol. Samples were resuspended in 50 mM triethylammonium bicarbonate. Total protein concentration was determined with a BCA kit (23227) from Thermo Fisher Scientific. Aliquots of each sample containing ∼100 μg of protein were brought to equal volumes with 50 mM triethylammonium bicarbonate buffer at pH 8. The mixtures were reduced with 20 mM DTT (37 °C for 1 h) and then alkylated with 40 mM iodoacetamide (30 min at RT in the dark). Samples were diluted 10-fold with 50 mM triethylammonium bicarbonate buffer at pH 8 and incubated ON at 37 °C with sequencing grade trypsin (Promega) at a 1:50 enzyme:substrate ratio (wt:wt). Peptide supernatants were collected and desalted with Oasis HLB 30-mg Sorbent Cartridges (Waters; 186003908), concentrated, and resuspended in a solution containing mass spectrometric “Hyper Reaction Monitoring” retention time peptide standards (Biognosys; Kit-3003) and 0.2% formic acid in water.
8
Analyzing Collagen VII Expression
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Transduced and untransduced cell pellets of umbilical cordederived mesenchymal stromal cells or 48-hour cultured serum-free supernatant supplemented with 50 mg/ml ascorbic acid and concentrated using ProteoExtract Protein Precipitation kit (MilliporeSigma, Burlington, MA) were resuspended in cell lysis buffer for total protein isolation. The total protein concentration in the supernatant was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and equal quantities (50 mg) of total protein were loaded on SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene fluoride membranes and incubated with anti-C7 antibody overnight at room temperature with shaking, followed by incubation with secondary antibody conjugated with horseradish peroxidase. Signal detection was performed using the ECL Plus system (GE Healthcare, Chicago, IL). Wild-type human fibroblasts were used to identify the full-length C7 band. Anti-vinculin monoclonal antibody (cl. V284, Sigma-Aldrich) was used as a total protein loading control for cell lysate samples. Total protein amounts in the samples from concentrated culture medium loaded on the SDS-PAGE were checked by Ponceau S (Sigma-Aldrich) staining of the polyvinylidene fluoride membrane following the protein transfer.
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