Iscove’s Modified Dulbecco’s Medium (IMDM), EpiLife™ Medium, Defined Keratinocytes SFM (K-SFM), mouse EGF recombinant protein (EGF), type IV collagenase, dispase, trypsin-EDTA, antibiotic-antimycotic and gentamicin were obtained from Life Technologies/Gibco (Grand Island, NY, USA). Pierce BCA Protein Assay Kit, TRIzol reagents, M-MLV reverse transcriptase, Fluo-4 AM, Pluronic F-127 and Pierce Peptide Desalting Spin Columns were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was bought from Hyclone (Pasching, Austria). Y-27632 was obtained from ATCC (Manassas, VA, USA). Taq DNA polymerase was purchased from TaKaRa (Tokyo, Japan). FASP Protein Digestion Kit (ab270519) and Phalloidin-iFluor 488 Reagent (ab176753) were purchased from abcam (Cambridge, UK). In Situ Cell Death Detection Kit (11684795910) was obtained from Roche Diagnostics (Indianapolis, IN, USA). DNAse I was from Amersham Biosciences (Piscataway, NJ, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
Fasp protein digestion kit
Manufactured by Abcam
Sourced in United States
The FASP Protein Digestion Kit is a tool designed for the efficient digestion of proteins prior to mass spectrometry analysis. It utilizes filter-aided sample preparation (FASP) technology to facilitate protein extraction, denaturation, reduction, alkylation, and digestion in a single device.
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Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Ziptip c18 tip, by Merck Group (2 mentions)
Trypsin, by Promega (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Pierce peptide desalting spin columns, by Thermo Fisher Scientific (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Cytiva (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Y27632, by American Type Culture Collection (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Phalloidin ifluor 488 reagent, by Abcam (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
2 n morpholino ethanesulfonic acid, by Merck Group (1 mentions)
11 protocols using fasp protein digestion kit
1
Isolation and Culture of Keratinocytes
2
Quantification of Cardiac Troponin I
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Human Cardiac Troponin Complex (cTn) NIST SRM 2921 was purchased from the National Institute of Standards and Technology (NIST) and used as a calibrant [26 (link)]. The cTnI-free human serum was obtained from Hytest Ltd. (Turko, Finland). SIL-protein, a monoclonal antibody against human cTnI (19c7) was obtained from Promise Proteomics (Grenoble, France). The SIL version of the monoclonal antibody’s peptide, SIL-DLPSPIER (> 95% purity 13C6, 15N4) was purchased from New England Peptide Inc (Gardner, MA, USA) as powder. Magnetic dextran nanoparticles (nanomag®-D), which particle diameters of 130 nm and the surface functionalities COOH for the covalent binding of the antibody, were obtained from Micromod Partikeltechnologie GmbH (Rostock, Germany). 1-Ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC), N-hydroxysuccinimide (NHS) and Pierce™ Trypsin/Lys-C Protease Mix, MS Grade were purchased from Thermo Scientific™ (Rockford, Illinois, USA). Phosphate Buffered Saline (PBS), Sodium azide and 2-(N-Morpholino) ethanesulfonic acid, 4-Morpholineethanesulfonic acid (MES), dithiothreitol (DTT) were purchased from Sigma-Aldrich (Darmstadt, Germany). FASP Protein Digestion Kit was obtained from Abcam (Boston, MA). Tris (Base) was purchased from Bioshop (Canada).
3
FASP-Based Protein Digestion for Mass Spectrometry
4
Protein Extraction and Identification
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For protein extraction, the trypsin digested whey fraction was mixed with Universal Protein Extraction (UPX) Kit (Expedeon-44101) and protease inhibitor cocktail (Thermo Sci.-87785). Samples were sonicated at 200 g and then boiled at 95 °C for 10 min. After the boiling procedure, samples were centrifuged for 10 min at 20,000 g. Peptide production was performed utilizing FASP Protein Digestion Kit (Expedeon-44250) and trypsin treatments (Pierce-90057) on the supernatant. Samples were diluted to 200 ng/μl with 0.1% formic acid. Detector and calibration settings were made with the MassLynx program (V4.1-Waters) that is specific to Xevo G2-XS Q-TOF-MS (Waters Corp, Milford, MA, USA) device where the analysis was carried out. The tryptic peptides were fractionated with an acetonitrile gradient (5–35%) in HSS T3 (Waters-186008818) column and analyzed by mass spectrometry upon electrospray ionization. Peptide data was identified in an m/z range of 50–1950. MS analysis was performed for 0.7 s and the data was collected from the entire peptide. UniProt protein database and Progenesis software were used for peptide identification.
5
Enterococcus faecalis Proteome Analysis
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The proteome analysis of the E. faecalis protein extracts by mass spectrometry and their corresponding data processing were carried out in a Thermo Scientific EASY-nLC 1000 nano-liquid chromatograph system/Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) at the CICT facilities of the University of Jaén in collaboration with the staff. For this, samples were digested in a solution following the protocol of the FASP protein digestion kit (Expedeon, Heidelberg, Germany) followed by a desalination according to the Pierce C18 Spin Columns protocol of Thermo Fisher Scientific. The results were compared with a specific database for Enterococcus faecalis in Uniprot.org with Proteome Discoverer 1.4 (Thermo Fisher Scientific, San Jose, CA, USA) and the search engine Sequest HT.
6
Protein Extraction and Digestion Protocol
7
FASP-Based Protein Digestion Protocol
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Three equivalent biological tissues were prepared by the FASP method using commercial kit (FASP Protein digestion Kit, Expedeon) following the supplier recommendations. Briefly, after the denaturation step previously described the denaturation buffer was removed by centrifugation at 14,000 g for 15 min. The sample was alkylated using 100 μL of 50 mM iodoacetamide in urea for 30 min in the dark, and excess alkylation reagents were eliminated by washing twice with 100 μL of 8 M urea solution (at 14,000 g for 15 min) and two more times with 50 mM ammonium bicarbonate (at 14,000 g for 15 minutes). The spin filters were then transferred to clean tubes and 50 μL of digestion solution containing trypsin was added to the filters. The protein to enzyme ratio was estimated to be 50:1. The top of the tubes were wrapped in parafilm to minimize evaporation and the tubes were placed for 4 h at 37 °C under 500 rpm agitation in a Thermomixer (Eppendorf). The peptides were concentrated to 20 μL using a speedvac vacuum concentrator (Thermo Scientific) and stored at −80 °C prior LC-MS/MS analysis (Fig. S1 ).
8
Tryptic Digestion and LC-MS/MS Analysis
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Proteins were digested using a FASP protein digestion kit (Expedeon). Briefly, 25 μg of proteins were alkylated with iodoacetamide and then digested with trypsin in 50 mM NH4HCO3 at 37 °C overnight. The digested peptides were recovered with the NH4HCO3 and 0.5 M NaCl, and dried using a SpeedVac concentrator (Thermo Fisher Scientific). The peptides were dissolved and separated on an analytical column (C18, 3 μm, 100 Å, 150 × 0.075 mm, AMR) by nano-flow HPLC system (AMR) in a linear gradient with 0.1% formic acid (A) and 0.1% formic acid in 90% acetonitrile (B) of 5–45% B in 140 min at flow rate of 250 nL/min. The eluted peptides were ionized in the nano-spray ion source, and detected by a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Mass and tandem mass spectra data were used for peptide identification with the SWISS-PROT database (Homo sapiens, 20,205 sequences in the Swiss Prot_2015_09.fasta file) and Mascot v.2.5.1 software (Matrix Science). The search parameters were as follows: tolerance of one missed trypsin cleavage; variable modifications on the methionine (oxidation, +16 Da), and serine, threonine, and tyrosine (phosphorylation, +80 Da); fixed modifications on cysteine (carbamidomethly, +57 Da); maximum precursor ion mass tolerance of ±10 ppm; and a fragment ion mass tolerance of ±0.8 Da.
9
Proteomics Analysis of Viral Infection
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As viral antigen was detected in lung tissue at 60 hpe in both bats and ferrets, this tissue was chosen to perform proteomics analysis for the detection of host and viral proteins. Lysate (400 μg of protein) from lung tissue of uninfected (0 hpe) and infected (60 hpe) animals was added to a Nanosep 10 kDa molecular weight cut-off filter (OD010C34; Pall) and processed using a filter-aided sample preparation (FASP) Protein Digestion Kit (Expedeon Inc) as described by manufacturers and by Wisniewski et al. [43 (link)]. All buffers were provided in the kit except for trypsin. Briefly, detergent was removed in a series of 8 M urea washes and cysteine residues were alkylated with the addition of iodoacetamide for 20 min in the dark. Proteins were washed twice with urea before being equilibrated with 50 mM ammonium bicarbonate. Proteomics grade porcine trypsin (T6567; Sigma) was added at a 1:80 (w/w) ratio of trypsin to protein and proteins were digested at 37°C overnight. The next day, tryptic fragments were eluted with the addition of 50 mM ammonium bicarbonate and 500 mM NaCl and particulates were removed by centrifugation at 16000 x g for 10 min. Each tissue sample was independently subjected to FASP tryptic digestion three times (experimental triplicates).
10
FASP-Based Protein Digestion for Mass Spectrometry
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Proteins from the micro-dissected cells were extracted and digested using the filter-aided sample preparation (FASP) protocol [23 ,26 (link)]. A commercial FASP Protein Digestion Kit (Expedeon Inc., San Diego, CA) was used in this work. The FASP procedure was performed as described previously [23 ,27 (link)]. Enzymatic digestion was performed by adding trypsin (Promega, Madison, MI) in 75 μL of 50 mM NH4HCO3 to the filter. The protein to enzyme ratio was 100:1. Samples were incubated overnight at 37°C. Released peptides were collected by centrifugation and desalted with ZipTip C18 tips (Millipore, Billerica, MA).
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