The serum (100 μL) collected from mice was diluted twice with 10% trichloroacetic acid and centrifuged at 9730×g for 15 min. The supernatants were collected and processed in 0.22 μM SPIN-X Centrifuge Tube Filters (Corning Costar) and used for the analyses. CT26 mock control cells and Arg1 OE cells (5 × 106) were cultured in 100-mm cell culture dishes for 24 h. After washing with PBS, the cells were collected and centrifuged at 2430×g for 5 min. Methanol (1 mL) was added to the cell pellets and incubated at room temperature for 10 min, and then H2O (0.5 mL) and chloroform were further added before centrifugation at 21,880×g for 15 min. The supernatants were collected and processed with 10-kDa centrifugal filter units (Millipore) and used for analysis. The prepared samples were analyzed with an amino acid analyzer (L-8900; Hitachi High-Technologies Corporation, Tokyo, Japan) in the Global Facility Center at Hokkaido University.
10 kda centrifugal filter unit
Manufactured by Merck Group
Sourced in United States
The 10-kDa centrifugal filter units are a laboratory device used for the separation and concentration of molecules based on their molecular weight. These filter units contain a semi-permeable membrane that allows the passage of molecules smaller than 10 kilodaltons (kDa) while retaining larger molecules during a centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Spin x centrifuge tube filter, by Corning (1 mentions)
L 8900, by Hitachi (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Ni nta sepharose, by GE Healthcare (1 mentions)
Hiload 16 600 superdex 200 gel filtration column, by GE Healthcare (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Protein g gravitrap, by GE Healthcare (1 mentions)
Nanodrop one uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using 10 kda centrifugal filter unit
1
Amino Acid Quantification in Cells
2
Extracellular HMGB1 Detection via Western Blot
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The supernatants of treated MH‐S cells were added to 10 KDa centrifugal filter units from Millipore Company for concentration and purification following the manufacturer's instructions. Western blot was used to detect extracellular HMGB1 level, and the volume of loading was determined by the number of cells.
3
Purification of Recombinant Proteins
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Purified His-tagged MYC (TP760019) was purchased from Origene Inc. GST-tagged TRIB3 (10731-H09B) and KDC domains, GST-tagged MAX (12885-H20B) and His-tagged MAX (12269-H08H) were purchased from Sinobiological Inc. The recombinant proteins UBE3B (His-tagged), UBE3B-C (His-tagged), UBE3B R346Q, and UBE3B C1036A were produced in FreestyleTM 293-F cells (Life Technology, Carlsbad, CA, USA) following transient transfection with 1 mg/mL DNA at a DNA/PEI ratio of 1:2.5 (PEI, Polyscience). Proteins were purified from culture supernatants using Ni-NTA-Sepharose (GE Healthcare) and dialyzed against PBS. Purified proteins were loaded onto a Hiload 16/600 Superdex 200 gel filtration column (GE Healthcare) to remove aggregates. Purified proteins were concentrated to 4 mg/mL by a 10-kDa Centrifugal Filter Unit (Millipore, Burlington, MA, USA) and filtered by a 0.22-μm Millipore filter.
4
Polyclonal IgG Production from Rabbits
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Polyclonal IgGs were developed in New Zealand White Rabbits (Eurogentec, Belgium) as per the company’s protocol. In brief, 0.8 mg of purified recombinantly P. vivax TRAg protein was injected in two rabbits (0.4 mg each) with 100 μg/immunisation/rabbit. Freund’s complete adjuvant was used for first immunisation followed by Freund’s incomplete adjuvant in booster doses given on days 14, 28 and 58. The final bleed was collected on day 87.
Total IgG was purified from the final bleed rabbit sera using Protein G Gravitrap (GE Healthcare, Cytiva). The sera were diluted as 1:5 in binding buffer (20 mM Sod. Phosphate, pH 7.4). The purification was done using Ab purification kit and the protocol was followed as per the kit’s manual. In brief, the diluted sera were loaded in the preequilibrated Gravitrap column and the eluant reloaded three times to enrich the binding and the beads washed with 10 ml of binding buffer. The IgG was finally eluted with 5 ml of elution buffer (0.1 M glycine-HCl, pH 2.7) poured into the column and collected directly in a 10 kDa centrifugal filter unit (Millipore) containing 0.225 ml of neutralisation buffer (1 M Tris-HCl, pH 9.0). The IgGs were buffer exchanged three times in RPMI 1640 media (Gibco) for any follow up assay. The final concentration of the purified IgG was determined using a Nanodrop one UV-Vis Spectrophotometer (Thermo Fisher).
Total IgG was purified from the final bleed rabbit sera using Protein G Gravitrap (GE Healthcare, Cytiva). The sera were diluted as 1:5 in binding buffer (20 mM Sod. Phosphate, pH 7.4). The purification was done using Ab purification kit and the protocol was followed as per the kit’s manual. In brief, the diluted sera were loaded in the preequilibrated Gravitrap column and the eluant reloaded three times to enrich the binding and the beads washed with 10 ml of binding buffer. The IgG was finally eluted with 5 ml of elution buffer (0.1 M glycine-HCl, pH 2.7) poured into the column and collected directly in a 10 kDa centrifugal filter unit (Millipore) containing 0.225 ml of neutralisation buffer (1 M Tris-HCl, pH 9.0). The IgGs were buffer exchanged three times in RPMI 1640 media (Gibco) for any follow up assay. The final concentration of the purified IgG was determined using a Nanodrop one UV-Vis Spectrophotometer (Thermo Fisher).
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