The treated cells were lysed immediately with the compatible lysis buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 600 mM guanidine HCl, 1% DDM, and protease inhibitor mixture (1 mM EDTA, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 μg/mL aprotinin). Protein concentration was determined by Pierce Micro BCA Kit (Thermo). The obtained tissue lysate was processed by using SISPROT protocol as previously described [17 (link)]. Briefly, the samples were firstly acidified to pH 2–3 and loaded onto 200 μL or 10 μL spintip device packed with one plug of C18 disk (3 M Empore, USA) and 0.6 mg of 20 μm POROS SCX beads (Applied Biosystems, USA) in tandem. Proteins were reduced by TCEP, alkylated by IAA and digested by trypsin (TPCK-treated, Sigma-Aldrich). The digested peptides were then transferred from the SCX beads to C18 disk with 200 mM ammonium formate (pH 10) and eluted from C18 disk with ACN concentration of 80% in 5 mM ammonium formate (pH 10).
Tpck treated
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TPCK-treated is a lab equipment product. It is a chemical compound used for various research and laboratory applications.
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6 protocols using tpck treated
1
Protein Extraction and Purification Protocol
2
Virus Titer Determination in MDCK Cells
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MDCK or Vero cells at 80 to 100% confluence seeded in 24-well plates were infected with the indicated viruses at an MOI of 0.1. After absorption for 1 h, the supernatant was removed and cells washed twice with 500 μl phosphate-buffered saline (PBS). Infected cells were overlaid with MEM containing 1 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) and incubated at 37°C. Supernatants were collected at the indicated time points and the virus titer determined by plaque assay in MDCK cells.
3
Assessing Accessibility of Reconstituted Peptides
4
Antiviral Effects of J. multifida Extracts
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To explore whether the extracts from the stems of J. multifida affected viral growth in pre-infected cells, MDCK cells were seeded in a 24-well plate (1 × 105 cells/well). The cells were infected with A/PR/8/34 (MOI; 0.001) in infection medium for 1 h at 37 °C in the presence of 5% CO2. The infected cells were washed prior to the addition of H2O, EtOAc, Hex, or CHCl3 J. multifida extracts (12.5 or 25 μg/mL in 0.5% DMSO) to the cells in infection medium supplemented with 3 μg/mL l -tosylamido-2-phenyl ethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich). DMSO (0.5%) and ribavirin (50 μM in 0.5% DMSO) were the negative and positive controls, respectively, for the inhibition on influenza A viral growth [18 (link)]. The cells were then incubated for 24, 48, or 72 h at 37 °C in the presence of 5% CO2. Cell culture media were collected from each well at predetermined time points. Viral titers (plaque forming units per mL [PFU/mL]) were determined as previously described [15 (link)].
5
Western Blot Analysis of Malaria Proteins
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Western blots of membrane proteins from pigmented trophozoite-IEs were performed using Triton-X 100-insoluble, SDS-soluble protein extracts of pigmented trophozoite-IEs as previously described [15 ]. Nitrocellulose membranes (Invitrogen) were probed with affinity-purified rat antibodies against recombinant RIF29 (1/2000) [74 (link), 75 (link)], rat antibodies against RIF40.2 (1/2000) [30 (link)], affinity-purified mouse antibodies against recombinant STEVOR3 protein (1/2000) [76 (link)], rabbit anti-SBP1 (1/1000) [20 (link)], rabbit anti-HSP70 antibodies (1/500; provided by Paul Gilson) and rabbit anti-aldolase (1/5000; provided by Jake Baum). Magnet-purified IEs were incubated with trypsin at 1 mg/mL (TPCK-treated, Sigma–Aldrich) or PBS as a negative control for 30 min at 37 °C, and the reaction stopped with trypsin inhibitor (Sigma–Aldrich) before the cells were extracted with SDS loading buffer, as previously described [30 (link)].
6
Accessibility of Reconstituted Peptides
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To determine the accessibility of LUV-reconstituted peptides, 20 μl proteoliposomes was incubated with 0.6 μg trypsin (TPCK-Treated, Sigma-Aldrich) at 37°C for 30 min.
Next, 95°C pre-heated SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02 % bromophenol blue) was added to stop the reaction. The samples were analyzed by SDS-PAGE and stained with Coomassie blue.
Next, 95°C pre-heated SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02 % bromophenol blue) was added to stop the reaction. The samples were analyzed by SDS-PAGE and stained with Coomassie blue.
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