The A549 cells were infected with H1N1 IAV (A/Beijing/501/2009) or H5N1 AIV (A/goose/Jilin/hb/2003). Phosphate‐buffered saline (PBS) was used to wash the cells three times. Viral infection was performed in infection medium. This comprised culture medium, lacking FBS, but containing TPCK‐trypsin (1 μg/ml) (Promega, WI, USA) and BSA (1.5%). The virus MOIs were 1, 0.1, or 0.01, depending on the assay. The cells were incubated for 60 min. and then washed with PBS. Infection medium was then added. The infected cells were cultured in 5% CO2 at 35°C.
Tpck trypsin
Manufactured by Promega
Sourced in United States
TPCK-trypsin is a protease enzyme derived from bovine pancreas. It is used to cleave peptide bonds at the carboxyl side of arginine or lysine residues in proteins and peptides.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Merck Group (1 mentions)
Acetonitrile acn, by Mallinckrodt (1 mentions)
Sonic dismembrator 500, by Thermo Fisher Scientific (1 mentions)
High performance streptavidin packed beads, by GE Healthcare (1 mentions)
Micro bio spin chromatography column, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Streptavidin sepharose bead, by GE Healthcare (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
6 protocols using tpck trypsin
1
Viral Infection Assay in A549 Cells
2
LCMS Protein Digestion Protocol
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Acetonitrile (ACN) and LCMS grade water were obtained from Mallinckrodt (Phillipsburg, NJ, USA). Tris, dithiothreitol (DTT) and formic acid (FA) were from Merck (Darmstadt, Germany). TPCK-trypsin was obtained from Promega Corporation (Madison, WI, USA). Tris(2-carboxyethyl) phosphine (TCEP) was from Fluka Chemie (Buchs, Germany). All other chemicals were of analytical grade.
3
Virus Titer Determination in MDCK Cells
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MDCK cells were plated in 96 well plates and grown overnight. The virus was serially 10-fold diluted and inoculated onto MDCK cell monolayers. After incubation at 37°C for 1 h, the virus suspensions were removed and replaced with serum-free DMEM containing 2 μg/ml TPCK trypsin (Promega, Madison, WI, USA). At 12, 24, 36, 48, 60, and 72 h.p.i., the cytopathic effect (CPE) was observed microscopically and the TCID50 value was calculated using the Reed-Muench method (LaBarre and Lowy, 2001 (link)).
4
Protease Interactor Identification via BioID-MS
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BioID-MS was conducted as described previously (42) . NLN or ClpP complementary DNA (cDNA) was fused in-frame with a mutant Escherichia coli biotin-conjugating enzyme, BirA R118G (or BirA*) into a tetracycline-inducible pcDNA5 FLP recombinase target/ tetracycline operator (FRT/TO) expression vector, which was then transfected into Flp-In T-REx HEK293 Flp-In cells. Cells were lysed, sonicated twice for 10 s at 35% amplitude (Sonic Dismembrator 500; Fisher Scientific), and centrifuged at 35,000g for 30 min at 4°C. Supernatants were passed through a Micro Bio-Spin chromatography column (Bio-Rad 732-6204) and incubated with 30 l of high-performance streptavidin packed beads (GE Healthcare) for 3 hours at 4°C on an end-over-end rotator. Beads were collected (2000 rpm, 2 min) and washed six times with 50 mM ammonium bicarbonate (pH 8.3). Beads were then treated with l-1-tosylamide- 2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Promega) for 16 hours at 37°C on an end-over-end rotator. After 16 hours, another 1 l of TPCK-trypsin was added for 2 hours and incubated in a water bath at 37°C. Supernatants were lyophilized and stored at 4°C for downstream MS analysis. Two biological and two technical replicates were completed for each protease. NLN's interactors were compared to ClpP's interactors, which were normalized to each protease's respective BirA* spectral counts.
5
Serum-free Culture Conditions for Proteomics
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AIM-V serum-free conditioning medium, RPMI 1640 medium, and fetal bovine serum were purchased from Invitrogen (Grand Island, Shanghai, China). Deuterium oxide (D2O) was purchased from Tenglong Weibo Technology (Qingdao, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer and 3-[(3-cholamidopropyl) dimethylamino]-1- propanesulfonate (CHAPS) were purchased from Sigma Aldrich (Sigma Aldrich, Shanghai, China). TPCK-trypsin was purchased from Promega (Promega, Shanghai, China). Acetonitrile (GC purity) was purchased from Merck (Merck, Shanghai, China). Lysis buffer, 4× separating gel buffer, and balanced salt solution were sterilized by passing through a 0.22-μm membrane filter before sub-packaging and storing at −80°C.
6
Affinity Purification of Bait Proteins
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Cell pellets were thawed and resuspended in 10 mL of modified RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor cocktail (Sigma-Aldrich), 1:100 Benzonase nuclease (Sigma-Aldrich)) at 4 °C (Coyaud et al.80 ). Cells were lysed by sonication for 30 s at 35 % power. Insoluble material was removed by centrifuging the lysate at 16,000 rpm for 30 min. The clarified supernatant containing the bait protein was then incubated with 30 µL of streptavidin-Sepharose beads (GE) at 4 °C for 3 h. The beads were washed six times with 50 mM ammonium bicarbonate pH 8.3 to remove non-specific binders. Peptides were generated by TPCK trypsin (Promega) digestion at 37 °C for 16 h. The flow-through containing the tryptic peptides was lyophilized and resuspended in 0.1% formic acid for LC-MS/MS.
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