For purification of recombinant protein by Heparin-Sepharose affinity chromatography, 1 ml of Heparin-Sepharose (GE Healthcare) was loaded in a glass column and equilibrated with 20 ml chromatography (C)-buffer (25 mM MES, pH 6.0, 100 mM NaCl, 2 mM CaCl2, and 1 mM NaN3). Diluted E200 (rmDNase1L2) or the FT (rmDNase1L3) of the DEAE-cellulose anion-exchange chromatography was run through the column followed by washing with 10 ml C-buffer. Elution occurred with 7.5 ml of C-buffer containing 1.5 M NaCl. The eluate was diluted 1:2 with C-buffer without NaCl and concentrated with a 3K filter unit for subsequent gel-filtration.
Heparin sepharose
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Heparin Sepharose is a chromatography resin used for the purification of proteins that interact with heparin, a highly sulfated glycosaminoglycan. It consists of heparin covalently coupled to a cross-linked agarose matrix. The resin can be used to capture and isolate a variety of heparin-binding proteins, including growth factors, enzymes, and coagulation factors.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Cht 2, by Bio-Rad (2 mentions)
Histrap hp, by GE Healthcare (2 mentions)
Dna plasmid isolation kits, by Qiagen (2 mentions)
Lysogeny broth, by Merck Group (2 mentions)
Dmem media, by Thermo Fisher Scientific (2 mentions)
Quikchange 2 xl mutagenesis kit, by Agilent Technologies (2 mentions)
Superdex 200, by GE Healthcare (2 mentions)
Uncoupled sepharose 4b, by GE Healthcare (2 mentions)
Nih3t3 cells, by American Type Culture Collection (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ni nta agarose, by Thermo Fisher Scientific (1 mentions)
Phospho erk1 2 p erk1 2, by Cell Signaling Technology (1 mentions)
Anti gst antibody, by Merck Group (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Anti human igg fc antibody coupled to hrp, by Abcam (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
27 protocols using heparin sepharose
1
Purifying Recombinant Proteins by Heparin-Sepharose
2
Purification of DRH-3, RIG-I, and MDA-5
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Full length DRH-3 was expressed and purified as previously described (15 (link)). The N-terminal deletion variant of DRH-3 (ΔN-DRH3) containing residues 300–1120 was cloned into the pET-SUMO expression vector and transformed into Rosetta II cells (Novagen). Cells were grown at 37°C to an Abs600 of 0.6 and induced overnight with 0.3 mM IPTG at 16°C. The ΔN-DRH3 construct was purified using essentially the same protocol as previously described for full length DRH3 (15 (link)). After concentration, ΔN-DRH3 was quantified by absorbance (ε280 = 79.19 mM−1 cm−1), flash frozen and stored at −80°C.
Full-length RIG-I was purified as previously described (11 (link)). Human MDA-5 was cloned into the pET-SUMO vector and transformed into Rosetta II cells (Novagen). Cells were grown at 37°C to an Abs600 of 0.6, and MDA-5 expression was induced with 0.5 mM IPTG at 16°C overnight. Like RIG-I, MDA-5 was purified using nickel affinity chromatography, followed by cationic exchange and size exclusion chromatography using Heparin Sepharose and HiLoad Superdex 200 columns (GE Healthcare). After concentration, MDA-5 was quantified by absorbance (ε280 = 92.71 mM−1 cm−1), flash frozen and stored at −80°C.
Full-length RIG-I was purified as previously described (11 (link)). Human MDA-5 was cloned into the pET-SUMO vector and transformed into Rosetta II cells (Novagen). Cells were grown at 37°C to an Abs600 of 0.6, and MDA-5 expression was induced with 0.5 mM IPTG at 16°C overnight. Like RIG-I, MDA-5 was purified using nickel affinity chromatography, followed by cationic exchange and size exclusion chromatography using Heparin Sepharose and HiLoad Superdex 200 columns (GE Healthcare). After concentration, MDA-5 was quantified by absorbance (ε280 = 92.71 mM−1 cm−1), flash frozen and stored at −80°C.
3
RIG-I Protein Purification Protocol
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The RIG-I gene was cloned in the protein expression vector pET28 SUMO and expressed as SUMO fusion proteins in Escherichia coli strain Rosetta (DE3) (Novagen). The protein was purified using a series of chromatography columns as published previously (3 (link)). The soluble lysate was fractionated through a HisTrap HP (GE Healthcare), followed by Ulp1 protease digestion to remove 6× His-SUMO tag, hydroxyapatite (CHT-II, Bio-Rad), and heparin Sepharose (GE Healthcare). The purified protein was dialyzed into 50 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, 5 mM DTT and 10% glycerol overnight at 4°C, frozen in liquid nitrogen and stored at –80°C.
4
Plasmid Isolation and Mutagenesis
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DNA plasmid isolation kits were purchased from Qiagen, USA and Quikchange II XL mutagenesis kits were obtained from Agilent. Competent cells (DH5α and BL-21(DE3)) were sourced from Novagen Inc., USA. Lysogeny broth (LB) was obtained from EMD Millipore, USA. Heparin sepharose was obtained from GE Healthcare, USA. VWR Scientific Inc, USA was the supplier for all buffer components including Na2HPO4, NaH2PO4 and NaCl. NIH 3T3 cells were obtained from ATCC and all the cell culture reagents including, DMEM media, fetal bovine serum (FBS) and penicillin–streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA).
5
Purification of Mlh1-Mlh3 Complexes
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Mlh1-Mlh3 and Mlh1-mlh3 mutant derivatives were purified from Sf9 cells infected with Bac-to-Bac baculovirus expression system using pFastBacDual constructs [16 (link)]. Mutant Mlh1-mlh3 complexes were purified using the same protocol developed to purify wild-type Mlh1-Mlh3. This involved the use of successive nickel-nitroloacetic acid-agarose (Qiagen) and heparin sepharose (GE Healthcare) column purifications. Mlh1-Mlh3 and mutant derivative yields were ~150 μg per 5 x 108 cells; aliquots from the final heparin purification were frozen in liquid N2 and stored at -80°C. Protein concentrations were determined by Bradford assay [96 (link)] using BSA standard. The mlh3-6, mlh3-32 and mlh3-45 mutations were introduced into pEAE358 (pPH-His10-MLH3-HA pFastBacDual construct; Rogacheva et al. [16 (link)]) by Quick Change (Stratagene). His10-mlh3-HA fragments were individually subcloned by restriction digestion into pEAE348 to form pFastBacDual constructs pEAE382 (Mlh1-mlh3-6), pEAE383 (Mlh1-mlh3-32) and pEAE384 (Mlh1-mlh3-45), in which the MLH1-FLAG gene is downstream of the p10 promoter and the His10-mlh3-HA gene is downstream of the pPH promoter. The sequence of the restriction fragments inserted into pEAE348 were confirmed by DNA sequencing (Cornell Biotechnology Resource Center). Msh2-Msh3 was purified as described previously [97 (link)].
6
FGFR and ERK1/2 Signaling Pathway Antibodies
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The primary antibodies directed against FGFR1 (#9740), FGFR2 (#11835), FGFR3 (#4574), phospho-FGFR1 (pFGFR1; #3476), ERK1/2 (#9102), and phospho-ERK1/2 (pERK1/2; #9101) were from Cell Signaling (Danvers, MA, USA). The anti-tubulin primary antibody (#T6557) and the anti-GST antibody (#G1160) were from Sigma-Aldrich (St. Louis, MO, USA). Anti-human IgG (Fc) antibody coupled to HRP (#ab97225) was from Abcam (Cambridge, UK). The primary antibodies directed against FGFR4 (#sc-136988) and the anti-His-Tag primary antibodies (#sc-8036) were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies were obtained from Jackson Immuno-Research Laboratories (Cambridge, UK). Protein A Sepharose, Glutathione Sepharose, and Heparin Sepharose resins were from GE Healthcare (Chicago, IL, USA). Ni–NTA agarose and PrestoBlue™ Cell Viability Reagent (Thermo Fisher Scientific, Waltham, MA, USA). α-Lactose Separopore resin was from bioWORLD (Irving, TX, USA).
7
Purification and Characterization of PC4 Variants
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Bacterially expressed recombinant N-terminal His6-tagged linker H1 variants (used for in vitro interaction studies) were subcloned in pET28b vector and purified using nickel-NTA (Ni-nitrilotriacetic acid) agarose (Millipore). Mutations in the N terminus region within the first serine rich tract of PC4 were introduced by using the site directed mutagenesis kit from Stratagene (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA, USA) as per the manufacturer's instructions. A series of phospho-deficient mutants generated upon mutating serine 13, 15, 17 and 19 in different combinations to alanine were labelled as MTP3, MTP5, MTP6, MTP7, MTP8 and MTP9 (Supplementary Figure S1B ). Bacterially expressed untagged recombinant PC4, phospho-mimic (PM-PC4) and phospho-deficient mutants of PC4 (MTPs) were expressed using pET11a vector and purified by heparin sepharose (GE Healthcare) and phosphocellulose P11 column chromatography (28 (link)). Recombinant core histones (Xenopus) H2A, H2B, H3 and H4, which come in inclusion bodies, were purified by denaturation in 8M urea followed by renaturation as described elsewhere (29 ). Purification of human linker histone H1was carried out as described elsewhere (30 (link)).
For cellular studies, Flag tagged Wild type PC4, PM-PC4 and MTP5 were subcloned in mammalian expression vector pFLAG-CMV-10.
For cellular studies, Flag tagged Wild type PC4, PM-PC4 and MTP5 were subcloned in mammalian expression vector pFLAG-CMV-10.
8
Purification of Recombinant β2GPI Protein
9
Purification of RIG-I Protein Constructs
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RIG‐I constructs were cloned into the protein expression vector pET28 SUMO and expressed as SUMO fusion proteins in Escherichia coli strain Rosetta (DE3) (Novagen). The proteins were purified using a series of chromatography columns, as published previously (Jiang et al, 2011 (link)). The soluble lysate was fractionated through HisTrap HP (GE Healthcare), followed by Ulp1 protease digestion to remove 6xHis‐SUMO tag, hydroxyapatite (CHT‐II, Bio‐Rad), and heparin sepharose (GE Healthcare). The purified protein was dialyzed into 50 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, 5 mM DTT, and 10% glycerol overnight at 4°C, frozen in liquid nitrogen, and stored at −80°C.
10
RNAP-Promoter DNA Open Complex Formation
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RNAP-promoter DNA open complexes (RPO) were formed as described previously (30 (link)–32 (link)). Briefly, 10 nM dsDNA was incubated with 50 nM E. coli RNAP holoenzyme in T8 buffer (50 mM Tris–HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 100 μg·ml−1 bovine serum albumin (BSA), 1 mM dithiothreitol, 5% glycerol) giving a final volume of 20 μl. The mixture was incubated at 37°C for 15 min, after which 1 mg/ml heparin sepharose (GE Healthcare) was added to disrupt non-specific RNAP–DNA complexes. The mixture was incubated at 37°C for 30 s and then centrifuged to remove sepharose beads. The supernatant was then removed and transferred to a pre-warmed Eppendorf tube and incubated for a further 20 min at 37°C.
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