Heparin agarose (GE Healthcare) was packed into a column and then equilibrated with 20 mM HEPES, pH7.4. Each of the purified recombinant proteins (HA-NL1-His wild-type and RA mutant, and LRRTM2-AP-Myc-His wild-type and RA mutant) was incubated with Heparin agarose overnight at 4°C. Bound protein was eluted from the column using a stepwise salt gradient of 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM and 1 M NaCl in 20 mM HEPES (pH 7.4). Eluates were collected for each step and probed using immunoblotting.
Heparin agarose
Manufactured by GE Healthcare
Heparin agarose is a chromatography medium used for the purification of proteins, enzymes, and other biomolecules. It consists of heparin, a sulfated glycosaminoglycan, immobilized on an agarose matrix. Heparin agarose exhibits a high affinity for proteins that interact with heparin, such as antithrombin, growth factors, and certain coagulation factors. The medium can be utilized in various chromatographic techniques, including affinity chromatography, to selectively bind and separate target molecules from complex mixtures.
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Lab products found in correlation
4 protocols using heparin agarose
1
Heparin Agarose Affinity Purification
2
NLGN1 Ectodomain Purification and Heparin Binding
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Purification of NLGN1 ectodomain proteins and the heparin binding assay were previously described (Zhang et al., 2018 (link)). Briefly, an N-terminal HA tagged and C-terminal His tagged NLGN1 ectodomain of each isoform was transiently expressed in HEK293T cells and purified with Ni-NTA agarose beads (QIAGEN). Purified NLGN1 ectodomains were incubated with heparin agarose (GE Healthcare) and eluted with NaCl in step-wise gradient concentrations (50 mM – 3 M) in 20 mM HEPES, pH 7.4. Collected eluates were analysed by immunoblotting with rat monoclonal HA antibody (Roche 11867431001; clone 3F10; 1:2000), secondary HRP-conjugated antibody, and detected using the SuperSignal Chemiluminescent kit (Thermo Fisher Scientific).
3
Purification of Ku70/80 Heterodimers
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Human DNA-PKcs and wild-type Ku70/80 proteins were purified as previously described (28 (link)). To purify Ku heterodimers with mutant Ku70, Sf9 cells were infected with Ku70 mutant and wild-type Ku80 baculoviruses and harvested 60 h post infection. The His6-tagged mutant Ku70 and His6-tagged wild-type Ku80 heterodimers were purified via Ni-NTA agarose (Qiagen), Superdex200 16/60 and Heparin agarose column chromatography (GE Life Sciences), as previously described (26 (link),28 (link)).
4
Purification of Bacterial UvrABC Proteins
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B. caldotenax UvrA, UvrB, and UvrC proteins were cloned into pET-28a(+) vector (Novagen) and expressed in E. coli BL21 (DE3) pLysS using standard protocol. Harvested cells were lysed using sonication, followed by centrifugation at 35,000 g to clear the lysate. Subsequently, all proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen), Heparin agarose (GE Healthcare), and size-exclusion chromatography using HiLoad 16/600 Superdex 200 (GE Healthcare). Cysteine residues for disulfide crosslinking (T251C or S91C) and a stop codon to generate domain 4 deletion construct (Δ594-658) were introduced to the UvrB expression vector using a QuikChange II site-directed mutagenesis kit (Agilent Technologies). To obtain homogeneous UvrB-DNA disulfide-crosslinked complexes, all native cysteine residues in B. caldotenax UvrB (C144, C211, and C303) were mutated to serine for both structural and biochemical studies. Only for crystallographic studies, His-tag was cleaved from UvrB by overnight incubation with TEV protease at 4°C prior to size-exclusion chromatography. UvrA and UvrC were frozen in 25 mM Tris pH 7.4, 250 mM NaCl, 20% (v/v) glycerol, and 5 mM βME at -80°C until use. UvrB was stored in the same buffer but was lacking βME.
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