MoFeP was desalted over a 10-DG gravity flow column (Bio-Rad) that had been equilibrated with a buffer solution (50 mM TRIS, pH 8.0, 500 mM NaCl) to remove DT. The protein was concentrated to ~50 μM in 30 kDa Microcon centrifugal filters (EMD/Millipore) and then oxidized with 2.5 mM IDS for 1 h. The protein solution was run over another 10-DG gravity flow column that had been equilibrated with a buffer solution (50 mM TRIS, pH 8.0, 500 mM NaCl) to remove IDS. The protein was concentrated to ~50 μM in 30 kDa Microcon centrifugal filters (EMD/Millipore), followed by addition of 1 mM metal (M = NiSO4, CoCl2, GaCl3, or 57FeSO4). After a 1-h incubation, excess metal was removed by exchanging the protein into a fresh buffer solution (50 mM TRIS, pH 8.0, 500 mM NaCl) with 30 kDa Microcon centrifugal filters (EMD/Millipore) and concentrated to ~50 μM. The first desalting and the IDS-oxidation steps were omitted during the preparation of the control EPR samples with reduced protein. All wt and βS188A MoFeP samples were prepared in parallel.
Microcon centrifugal filter
Manufactured by Merck Group
Sourced in United States, Germany
Microcon centrifugal filters are laboratory equipment used for sample concentration and purification. They function by applying centrifugal force to separate sample components based on their molecular weight or size.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear extraction kit, by Thermo Fisher Scientific (5 mentions)
Gel filtration calibration kit hmw, by GE Healthcare (5 mentions)
Superose 6 size exclusion column, by GE Healthcare (4 mentions)
Stratagene uv stratalinker 2400, by Corning (2 mentions)
U shaped bottom microplates, by Corning (2 mentions)
Drx 500 spectrometer, by Bruker (2 mentions)
Biotin protein labeling kit, by Roche (1 mentions)
Ab192778, by Abcam (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
α tubulin t9026, by Merck Group (1 mentions)
Cthrc1, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Caco 2 htb 37, by American Type Culture Collection (1 mentions)
Cm5 dextran sensor chip, by GE Healthcare (1 mentions)
Silica gel 60 tlc plate, by Merck Group (1 mentions)
40 protocols using microcon centrifugal filter
1
Purification and Metal Incorporation of MoFeP
2
Generation of SLC26A6-expressing FRT Cell Line
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A vector containing (murine) slc26a6 cDNA was purchased from Origene (cDNA clone MC202861) and confirmed by sequence analysis. The slc26a6 cDNA was excised by digestion with SpeI and NotI and subcloned into the pLVX-IRES-mCherry lentiviral expression cassette (Clontech) at NheI and NotI sites. To generate a cell line for screening, slc26a6 lentiviral particles were produced in HEK293 cells using the pRSV-Rev, pMDLg/pRRE, and pMD2.G packaging vectors (Addgene, deposited by Didier Trono, plasmids 12253, 12251, and 12259) and used to transduce Fischer rat thyroid (FRT) cells previously transduced with lentivirus expressing EYFP-H148Q/I152L/F46L (YFP) (7 (link)). FRT cells were obtained from the University of California, San Francisco, Cystic Fibrosis Drug Discovery Core Center. For lentivirus production, HEK293T cells (ATCC, CRL-3216) were transfected with pLVX-IRES-mCherry-slc26a6, pRSV-Rev, pMDLg/pRRE, and pMD2.G using NanoFect transfection reagent (Alstem) per the manufacturer’s instructions. After 1 day, the medium serum content was reduced to 2.5%, and after 2 days, the medium was harvested, centrifuged (5 min, 5000g) and concentrated using Microcon centrifugal filters (MilliporeSigma) with 10-kDa molecular-weight cutoff.
3
Biotin Labeling of Recombinant MSP-1
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Biotin was linked to MSP-1 using a Biotin Protein Labeling Kit (Roche) following the manufacturer's instructions. The purified recombinant MSP-1 protein solution was subjected to buffer exchange with PBS by centrifugation using Microcon centrifugal filters (10K MWCO, Millipore Sigma). Based on the protocol, the molar ratio between a 50 kDa protein and Biotin-7-NHS (D-Biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester, MW = 454.5) is 1:10. Therefore, about 1 mg of purified recombinant MSP-1 protein was added to 1 mL PBS, followed by the addition of 0.09 mg freshly prepared Biotin-7-NHS (D-Biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester). The mixture was incubated at room temperature for 2 h with gentle agitation to facilitate free amino groups of MSP-1 to react with D-Biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester (biotin-7-NHS) and form stable amide bonds. The reaction mixture was then applied to a pre-prepared Sephadex G-25 column to remove the residual non-reacted biotin-7-NHS by gel filtration. After letting the reaction mixture flow through the column, the biotin-labeled protein was eluted with 3.5 mL PBS and stored at 4 °C until use.
4
Generating HLA-A*02:01 Monomers from E. coli
5
Recombinant HLA-A*02:01 Monomer Production
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To generate HLA-A*02:01 (MHC-I) monomers, the corresponding α-chain and β2m protein constructs were overexpressed separately in E. coli. The protein was refolded from the inclusion bodies in the presence of an ultraviolet (UV)–cleavable peptide and biotinylated for downstream applications as described previously (63 (link)). After purification, the protein was concentrated and stored with 20% glycerol at −80°C. For each epitope specificity tested in this study, peptide exchange reactions were set up in a volume of 100 μl containing 0.2 mM peptide and HLA-A*02:01 protein (100 μg/ml) in PBS (pH 7.4). The reaction mixture was exposed to 365 nm UV light irradiation for 20 min using a Stratagene UV Stratalinker 2400 in 96-well U-shaped bottom microplates (Corning). The plate was then transferred to 4°C overnight to complete the exchange. The protein was subsequently buffer-exchanged against PBS (pH 7.4) using Microcon centrifugal filters (10-kDa cutoff, MilliporeSigma) to remove the excess free peptide and subsequently spun at 13,000g for 15 min at 4°C to remove aggregates. The protein was filtered and stored at 4°C until further use.
6
Bispecific IgG1 Antibody Generation
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Example 3
Bispecific IgG1 antibodies were generated by Fab-arm-exchange under controlled reducing conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011/131746. The F405L and K409R (EU numbering) mutations were introduced in CD37 antibodies to create antibody pairs with complementary CH3 domains. The F405L and K409R mutations were in certain cases combined with E430G mutation.
To generate bispecific antibodies, the two parental complementary antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE at 31° C. for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30 k, Millipore) according to the manufacturer's protocol.
7
Protein Expression Analysis of Cancer Signaling
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Whole cell lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo, USA) and culture supernatants were concentrated using Microcon centrifugal filters (Millipore, USA). Western blot was performed as previously described [17 (link)]. Primary antibodies against the following proteins were used: CTHRC1 (ab192778, Abcam, USA), p-c-Raf (9427, CST, USA), p-MEK1/2 (9154, CST, USA), p-ERK1/2 (4370, CST, USA), ERK1/2 (4695, CST, USA), p-FRA-1 (5841, CST, USA), FRA-1 (5281, CST, USA), cyclinD1 (2978, CST, USA), snail1 (3879, CST, USA), and MMP14 (13130, CST, USA). α − Tubulin (T9026, Sigma-Aldrich, USA) was used as a loading control.
8
Quantifying 2-5A Levels in Transfected Cells
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Hey1B cells were plated at 4 × 105 cells/well in six-well plates for 20 h. Plasmid DNAs (1 µg per well) of the empty vector (pCAGGS), AKAP7CD, AKAP7, and Flag-tagged-AKAP7H93A;H185R were transfected using Lipofectamine 2000. After 16 h, cells were mock transfected or transfected with poly(rI) ⋅ poly(rC) (pIC) (EMD Biosciences) at 1 µg/well for 3 h. The cells were washed with phosphate-buffered saline (PBS), lysed in buffer (50 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 1% NP-40, 200 µM sodium orthovanadate, 2 mM EDTA, 5 mM MgCl2, 5 mM DTT) and heated to 95°C for 7 min. Lysates were centrifuged for 10 min at 14,000 × g at room temperature, and supernatants were passed through Microcon centrifugal filters with a molecular mass cutoff of 3 kDa (Millipore Corporation) for 45 min at 11,000 × g. Levels of 2-5A were determined by RNase L-based FRET assays in comparison to a standard curve of authentic (2′-5′)p3A3 as we described previously (20 (link)).
9
Deuterium Exchange of LDL Subtypes
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LDL samples were concentrated to approximately 10 mg/ml using Microcon centrifugal filters (Millipore) and dialyzed in a Novagen TM Dialyzer D-Mini Tubes (Merck, Darmstadt, Germany) to exchange the sample buffer by deuterated buffer. The dialyses were carried out at 4°C in a closed container in order to prevent rehydration. The H2O-D2O exchange was followed by the analysis of infrared spectrum of withdrawn buffer in the same conditions as samples were measured later. The D2O buffer was restored for several times until no change was detected as a result of being in contact with the samples. There had been 19 analyzed samples in the study. Each sample was measured by triplicate and in different days to corroborate that there were no errors caused by the methodology. From the 19 analyzed samples, 4 were assigned to A subtype, 9 to B subtype and 6 to C subtype (see Results section and Table S1 ). To avoid an incorrect interpretation of such frequencies as the ones naturally occurring in population, seven samples were arbitrarily excluded from Table 1 (see Results section) thus showing equal number of each subtype.
10
Investigating LPS-induced Inflammation
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CNBr-activated sepharose 4 fast flow resin and CM5 dextran sensor chip were from GE healthcare (Piscataway, NJ). Lipopolysaccharide (LPS, E. Coli. 0111:B4), Recombinant human histone H3, triton X-100 and protease inhibitors were purchased from Sigma (St. Louis, MO). Dextran sulfate sodium (MW = 36,000–50,000 Da) was purchased from MP Biomedicals (Solon, OH). RAW 264.7 (TIB-71), HELA (CCL-2) and Caco-2 (HTB-37) cells were obtained from American type culture collection (Rockville, MD). Human TNF and anti-human TNF antibody were purchased from R & D system Inc. (Minneapolis, MN). The lactate dehydrogenase (LDH) cytotoxicity kit was from Takara Inc., (Otsu, Shiga, Japan). HMGB1 ELISA kit was from IBL international (Hamberg, Germany). QIAzol lysis reagent was from Qiagen (Valencia, CA). One step RT-PCR kit was obtained from Eurogentec (Fremont, CA). Poly I∶C was from InvivoGen (San Diego, CA). Light Cycler 480 instrument was purchased from Roche Diagnostics (Munich, Germany). RNase free DNase I and anti-histone H3 antibody were purchased from Thermo Scientific (Rockford, IL). Superscript III First-strand Synthesis System for cDNA synthesis was purchased from Invitrogen (Grand Island, NY). Microcon centrifugal filters were from Millipore (Billerica, MA).
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