His bind resin
Manufactured by Merck Group
Sourced in Germany, United States
His-Bind resin is a nickel-charged iminodiacetic acid (Ni-IDA) agarose-based resin designed for the purification of recombinant proteins containing a histidine-tag. It is used to capture and purify these tagged proteins from complex mixtures, such as cell lysates or culture supernatants.
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Lab products found in correlation
Bca protein assay kit, by Beyotime (4 mentions)
Pet19b, by GenScript (3 mentions)
Incomplete freund s adjuvant, by Merck Group (3 mentions)
Complete freund s adjuvant, by Merck Group (3 mentions)
Lysozyme, by Merck Group (3 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (2 mentions)
Overnight express instant tb medium, by Merck Group (1 mentions)
Ab817, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Clone ha 7, by Merck Group (1 mentions)
As05086, by Agrisera (1 mentions)
Analytic selector kit, by Anatrace (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Glutathione sepharose 4 fast flow beads, by GE Healthcare (1 mentions)
D glucose, by Cambridge Isotopes (1 mentions)
Anti flag m2 agarose bead, by Merck Group (1 mentions)
Anti c myc agarose beads, by Merck Group (1 mentions)
Bl21 de3, by New England Biolabs (1 mentions)
E toxate kit, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
24 protocols using his bind resin
1
Affinity Purification of A. phagocytophilum
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A. phagocytophilum DC bacteria were recovered from 2 × 107 heavily infected (≥90%) HL-60 cells by sonication as described previously (69 (link)). DC organisms were incubated with a 50-μl suspension of His-Bind resin (MilliporeSigma) in the presence or absence of 4 μg of His-PDI in a final volume of 1.1 ml PBS at 4°C for 3 h with constant rotation. The resin was pelleted by centrifugation at 1,000 × g for 1 min at 4°C, followed by the addition of 100 μl of lysis buffer and incubation on ice for 30 min. The resin was washed three times with 1 ml lysis buffer each time with centrifugation at 1,000 × g for 1 min at 4°C followed by resuspension in Laemmli buffer with freshly added β-mercaptoethanol. Eluates and input lysates were examined by Western blot analysis.
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2
Recombinant Ovalbumin and Lhc1 Purification
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Ovalbumin (Ova) and Lhc1 were synthesized and cloned in pET19b by Genscript (Piscataway, NJ, USA). Recombinant proteins were expressed in E. coli and purified using His·Bind resin (MilliporeSigma) as previously described (10 (link)). Briefly, BL21 E. coli containing recombinant protein-expressing plasmid were inoculated into Overnight Express Instant TB medium (MilliporeSigma) from glycerol stocks maintained at -80°C. Cells were lysed and soluble material was purified over a His·Bind nickel column, with all purification buffers containing 6M urea to maintain protein solubility. Eluted fractions were selected based on SDS-PAGE analysis and pooled for dialysis to remove imidazole. Dialyzed protein was concentrated to 10 mg/mL, and protein stocks were stored at -80°C.
3
Antibody Generation and Characterization
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All custom made antibodies were prepared in rabbits by Eurogentec (Eurogentec SA). Rabbit antisera were produced against DRM2 peptides EP112214 (NSDDEKDPNSNENGS) and EP112215 (ESKGEPRSSVDDEPI) following their double‐X immunization program and then affinity‐purified on EP112215. Antibodies for NRPD1 detection were also raised in rabbits against EP112201 (ESKGEPRSSVDDEPI) peptide and affinity purified by Eurogentec. His‐tagged UAP56 protein was produced from pET‐28a‐UAP56 in BL21 E. coli strain and purified with His‐bind resin following the supplier's instructions (Millipore). Anti‐UAP56 serum was then produced in rabbits using this recombinant protein as antigen. Anti‐AGO4 antibodies were previously used by Lahmy et al. 19 . Monoclonal antibody 8WG16 (ab817; Abcam) was used to detect NRPB1; histone H3 (ab1791; Abcam) and UGPase polyclonal antibodies (AS05 086, Agrisera) were also used for nucleus and cytoplasm controls. Affinity‐purified anti‐HA antibodies coupled to HRP (Sigma‐Aldrich, clone HA‐7) were used to detect DRM2 in transgenic tagged lines.
4
Differential Filtration Assay for VanS_A
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The differential filtration assay was performed as described by Vergis et al. [33 (link)], using the Analytic Selector kit (Anatrace). Briefy, 500 μg of full-length VanSA was captured using a 20% slurry of His-bind resin (Millipore) in wash buffer (50 mM Tris, 500 mM NaCl, 40 mM imidazole pH 7.7) + 0.1% DDM. The slurry was dispensed into each well of a 0.2 μm filter plate, after which the beads were washed with wash buffer + 0.1% DDM, and then detergent-exchanged into the new detergents from the Analytic Selector kit. The protein was eluted from the His-bind beads and then applied directly to either a small (100 kDa) or large (300 kDa) MWCO filter plate. The eluates from these filtrations were analyzed by dot blot using an anti-6xHis antibody (Proteintech # HRP 66005); blots were scanned using a LI-COR Odyssey-FC imaging system.
5
Recombinant Protein Expression and Purification
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Recombinant protein expression constructs were transformed into various E. coli strains for optimum expression (table S2). Protein samples used for EMSA studies were expressed in standard LB media. Proteins used for acquiring 15N HSQC NMR spectrum were expressed in M9 minimal media, with 15N-labeled ammonium chloride (15N, 98%+) (Cambridge Isotope Laboratories Inc.) as nitrogen source and d -glucose (Cambridge Isotope Laboratories Inc.) as carbon source. Protein expression was induced by isopropyl-β-d -thiogalactopyranoside. TrxA-His6–tagged proteins were purified with a His•bind resin (Millipore) according to the manufacturer’s protocol. GST-tagged proteins were purified with Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) according to the manufacturer’s protocol. For protein samples used for acquiring the 15N HSQC NMR spectrum, the fusion tag was removed by 3C protease. Purified proteins were dialyzed in dialysis buffer [20 mM Hepes (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 15% glycerol, and 1 mM dithiothreitol (DTT)] at 4°C overnight.
6
Integrin Tail Precipitation Assay
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Recombinant His-tagged integrin tails (Mark Ginsberg, UCSD) were produced and purified as previously described72 (link),73 (link) then immobilized on His-bind resin (Millipore, Billerica, MA ). HeLa cells were transfected with the indicated constructs then lysed in XTT lysis buffer. Cell lysates were then incubated with integrin tails for 24 h, followed by boiling in SDS-PAGE loading buffer. Precipitated PEA-15 was detected by Western blot, and integrin tail expression was detected by coomassie stain.
7
Co2+ Binding Assay for RGS4/5
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To measure the Co2+ binding of RGS4/5, ADO-deficient SH-SY5Y cell lysates were incubated with HisBind resin (Millipore 69,670) pre-charged with Co2+ ions, allowing the precipitation of Co2+-binding proteins as described previously (29 (link)). 25 μl (packed volume) of HisBind resin was washed in PBS and then incubated with 50 mM CoCl2 at room temperature for 1 h, then washed five times with lysis buffer. Cell lysate (15 cm dish per condition) was then incubated with Co2+ (or control) loaded resin for 1 h under constant rotation at 4 °C. Following this, the resin was pelleted (2000 rpm, 2 min) and a sample was taken and mixed with sample buffer to represent an unbound fraction. The resin was then further washed and subjected to the following elutions; 60 μl 60 mM imidazole for 10 min, 60 μl 1 M imidazole, then finally 70 μl SDS sample buffer. After each elution, the supernatant was taken and heated at 95 °C for 5 min, then subjected to SDS-PAGE and immunoblotted as described above.
8
RGA-TAP Purification and Characterization
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RGA-TAP was tandem affinity-purified from the 35S:RGA-TAP rga-24 ga1-3 transgenic lines in the wild-type SEC and SPY backgrounds (labeled as “WT”) (Zentella et al. 2007 (link)) or the spy-8 and sec-3 backgrounds using His-Bind resin (EMD Millipore) followed by anti-cMyc-agarose beads (Sigma-Aldrich). His-Flag-RGA protein transiently expressed in tobacco was tandem affinity-purified using a His-Bind resin followed by anti-Flag-M2-agarose beads (Sigma-Aldrich). Detailed protein purification procedures and GalT assays are provided in the Supplemental Material.
9
Recombinant Chitin Deacetylase Proteins
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The National Center for Biotechnology Information file for C. neoformans var. grubii H99 strain (taxid:235443) served as the source for cDNA and protein sequences of Cda1 (CNAG_05799), Cda2 (CNAG_01230), Cda3 (CNAG_01239), and Fpd1 (CNAG_06291). cDNAs for these proteins and the mutated versions of Cda2 (Cda2-M1 and Cda2-M2) were synthesized and cloned into pET19b (GenScript) so that the vector-encoded His tag was integrated with the N terminus of the cDNA. Recombinant protein was made in E. coli strain BL21(DE3) (New England BioLabs) using Overnite Express TB medium (Novagen) and purified on His·Bind resin (EMD Millipore) in the presence of 6 M urea, as described previously (16 (link)). Following elution with imidazole, proteins were dialyzed against 6 M urea/20 mM Tris-HCl, pH 7.9, and concentrated to 10 mg/mL using Amicon Ultra-15 centrifugal filters (10-kDa cutoff; Merck Millipore). The protein concentration was determined by the bicinchoninic acid (BCA) assay. To assess purity, the recombinant proteins were resolved by SDS-PAGE and stained with Coomassie InstantBlue (Expedeon, Ltd.).
10
Purification of Polo-Like Kinase Domains
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Human PLK1, PLK2 and PLK3 PBDs were expressed as previously described [33] (link). Gene sequences were amplified and cloned into a modified pET28a vector (PLK1) or into a modified pQE70 vector carrying a C-terminal 6× His tag and an N-terminal MBP tag (PLK2 and PLK3). Plasmids were transfected into Novagen Rosetta BL21DE3 cells (Merck, Darmstadt, Germany) for protein expression. Then, His•Bind® Resin (Merck) was used for protein purification and dialysis into buffer containing 50 mM Tris (pH 8.0) 200 mM NaCl (Plk1: 400 mM NaCl), 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.1% Nonidet P-40.
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