Nhs activated sepharose

Manufactured by GE Healthcare

Sourced in United States, Germany

NHS-activated Sepharose is a pre-activated agarose-based resin designed for covalent immobilization of proteins and other ligands containing primary amino groups. It provides a simple and effective method for coupling ligands to the resin, facilitating the development of affinity chromatography applications.

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NHS-activated Sepharose 4 Fast Flow (18 citations) NHS-Sepharose (8 citations) NHS-activated Sepharose 4 fast flow beads (8 citations) NHS-activated Sepharose beads (7 citations) NHS-activated Sepharose Fast Flow (3 citations) NHS-activated sepharose 4 fast flow resin (3 citations) NHS-activated Sepharose 4B (2 citations) HiTrap NHS-activated Sepharose High Performance column (2 citations) NHS-activated sepharose 4 Fast Flow column (2 citations) HiTrap NHS‐Activated HP Sepharose column (2 citations)

21 protocols using nhs activated sepharose

Polyclonal Anti-CPn0809 Antibody Generation

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For generation of a polyclonal anti-CPn0809 antibody, purified recombinant His₆-CPn0809 (aa 1–253) protein was used to immunize two rabbits. Immunization was performed first with purified protein cut from a SDS gel and subsequently with native soluble protein. Immunizations were carried out by Eurogentec (Belgium).
The polyclonal anti-CPn0809 antibody was antigen-purified against GST-CPn0809N-His₆ protein coupled to NHS-activated Sepharose (GE Healthcare Life Sciences) following the manufacturer’s instructions and a standard protocol [35 ].

Engel A.C., Herbst F., Kerres A., Galle J.N, & Hegemann J.H. (2016). The Type III Secretion System-Related CPn0809 from Chlamydia pneumoniae. PLoS ONE, 11(2), e0148509.

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Affinity Purification of PDZ Proteins

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A synthetic peptide corresponding to the C-terminus of GKAP/SAPAP1 (sequence IYIPEAQTRL) was obtained from Genemed Synthesis (San Antonio, USA) and coupled to NHS-activated sepharose (GE Healthcare) at a concentration of 3 mg/ml matrix. PDZ domain containing proteins were expressed in human embryonic kidney (HEK293) cells by calcium phosphate transfection of expression vectors. Cells were lyzed in RIPA buffer, followed by centrifugation at 20.500× g. PDZ fusion protein was precipitated from cleared lysates using the immobilized peptide and analyzed by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue; bands were cut out, digested with trypsin, and analyzed by mass spectrometry as described [25] (link).

Studtmann K., Ölschläger-Schütt J., Buck F., Richter D., Sala C., Bockmann J., Kindler S, & Kreienkamp H.J. (2014). A Non-Canonical Initiation Site Is Required for Efficient Translation of the Dendritically Localized Shank1 mRNA. PLoS ONE, 9(2), e88518.

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Immobilizing Compounds on Sepharose Beads

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Compounds were immobilized on sepharose beads through covalent linkage using their primary amino groups [62 (link)]. NHS-activated sepharose (GE Health-care, Freiburg, Germany) and the compounds were equilibrated in DMSO. Coupling densities of compounds included in KBγ were adjusted as previously described [28 (link)], linkable dasatinib was adjusted to a coupling density of 2 μmol per ml beads. Fifteen microliters of triethylamine was added to 1 ml beads to start the coupling reaction, and the mixture was incubated on an end-over-end shaker for 16–20 h in the dark. Free NHS groups on beads were blocked by adding 50 μl of amino ethanol and incubating on an end-over-end shaker for 16–20 h in the dark. Coupled beads were washed and stored in ethanol at 4°C in the dark. The coupling reaction was monitored by LC-MS.

Montenegro R.C., Howarth A., Ceroni A., Fedele V., Farran B., Mesquita F.P., Frejno M., Berger B.T., Heinzlmeir S., Sailem H.Z., Tesch R., Ebner D., Knapp S., Burbano R., Kuster B, & Müller S. (2020). Identification of molecular targets for the targeted treatment of gastric cancer using dasatinib. Oncotarget, 11(5), 535-549.

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UIML2 Domain Coupling to NHS-Sepharose

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The purified UIMLx2 domain was eluted and then resuspended in coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl pH 8.3) before being coupled to NHS-activated Sepharose (GE Healthcare) following the manufacturer’s instructions. The UIML2-conjugated agarose was stored at 4 °C in PBS supplemented with 30% glycine (36 (link)).

Villamil M., Xiao W., Yu C., Huang L., Xu P, & Kaiser P. (2021). The Ubiquitin Interacting Motif-Like Domain of Met4 Selectively Binds K48 Polyubiquitin Chains. Molecular & Cellular Proteomics : MCP, 21(1), 100175.

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Antibody Raised Against Aiptasia LAMP1 Homolog

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An antibody against the Aiptasia LAMP1 homolog (LOC110235349) was raised against the peptide IIGRRKSQRGYEKV (KXJ16564.1) coupled to the adjuvant keyhole limpet hemocyanin in rabbits (BioScience GmbH). The antibody was affinity purified from the third bleed using the synthetic peptide coupled to N-hydroxysuccinimide esters (NHS)-activated sepharose (17090601, GE Health Care Life Sciences) according to manufacturer’s protocols.

Jacobovitz M.R., Rupp S., Voss P.A., Maegele I., Gornik S.G, & Guse A. (2021). Dinoflagellate symbionts escape vomocytosis by host cell immune suppression. Nature microbiology, 6(6), 769-782.

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Antibody Glycoprofiling Mass Spectrometry

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2-Aminobenzoic acid, ethanolamine, formic acid, picoline borane, DMSO, ¹³C aminobenzoic acid were from Sigma (Munich, Germany). PNGaseF was from Roche (Penzberg, Germany). Acetic acid, acetonitrile and hydrochloric acid were from Merck (Darmstadt, Germany). NHS activated sepharose, Sephadex® G-10 96-well plates and 96-well deep well plates were from GE Healthcare (Munich, Germany). Antigen was from Peprotech (Hamburg, Germany). Phosphate buffered saline was from Gibco/Life technologies (Darmstadt, Germany).
Multicreen THS HV filter plates were from Milipore. IdeS protease was Genovis (Lund, Sweden). 96-well plates were from Nunc/Thermo Scientific (Munich, Germany). AcroPrep™ Advance Omega™ 10K 96-well filter plates were from Pall (Dreieich, Germany). Preclinical rabbit serum samples were obtained from clinical bioanalytics at Sandoz.

Higel F., Seidl A., Demelbauer U., Viertlboeck-Schudy M., Koppenburg V., Kronthaler U., Sörgel F, & Frieß W. (2015). N-glycan PK Profiling Using a High Sensitivity nanoLCMS Work-Flow with Heavy Stable Isotope Labeled Internal Standard and Application to a Preclinical Study of an IgG1 Biopharmaceutical. Pharmaceutical Research, 32(11), 3649-3659.

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Recombinant Galectin Immobilization Protocol

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Recombinant LGALS-11 and LGALS-14 were expressed and purified as described previously (Sakthivel et al., 2015 (link); Fig. 1). The recombinant protein (5 mg/ml) was buffer-exchanged into HEPES buffer (10 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 10 mM TCEP) and immobilised by coupling to N-hydroxysuccinamide (NHS)-activated Sepharose (GE Healthcare, Chicago, IL, USA) following the manufacturer’s protocol. Briefly, 4 ml of NHS-activated Sepharose was washed with 15 column-volumes of ice-cold 1 mM HCl. The washed Sepharose beads were equilibrated with 20 ml of coupling buffer (10 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 10 mM TCEP). Following equilibration, LGALS-11 and LGALS-14 were added separately to the activated Sepharose and allowed to couple for 5 h at 22 °C. Following the coupling reaction, the unused, activated sites were blocked using 15 column-volumes of blocking buffer (100 mM Tris–HCl pH 8.0, 100 mM NaCl) for 3 h. Following blocking, the Sepharose beads were washed alternatively six times with 15 column-volumes of 100 mM Tris-HCL pH 8.0 followed by 100 mM sodium acetate pH 5.0 and then 250 mM NaCl. The galectin affinity column was maintained in storage buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 10 mM TCEP, NaAc 0.02% (w/v)) until further use. A control resin was also prepared without any protein ligand.

Sakthivel D., Swan J., Preston S., Shakif-Azam M., Faou P., Jiao Y., Downs R., Rajapaksha H., Gasser R., Piedrafita D, & Beddoe T. (2018). Proteomic identification of galectin-11 and 14 ligands from Haemonchus contortus. PeerJ, 6, e4510.

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Amine-Functionalized DFHO Sepharose Coupling

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Amine-functionalized DFHO was first dissolved in DMSO at a concentration of 40 mM and then diluted into 100 mM HEPES buffer pH 7.5 with a final concentration of 5% DMSO and 2 mM amine-functionalized DFHO. This fluorophore solution was then added to NHS-activated Sepharose (GE Life Sciences), which had been preequilibrated with 2 volumes of ice-cold buffer. The resin was then incubated with amine-functionalized DFHO solution overnight at 4°C in the dark. The resin was washed with reaction buffer and incubated with 100 mM Tris pH 8.0 for 2 h at 25°C to react with any remaining NHS-activated sites. After thorough washing, the resin was stored in 1:1 ethanol:100 mM sodium acetate pH 5.4 at 4°C. The efficiency of sepharose coupling was monitored by measuring the absorbance at 400 nm of free DFHO in the flow-through. Using this approach, we estimate that the resin contains approximately 5 μmol of fluorophore per ml.

Song W., Filonov G.S., Kim H., Hirsch M., Li X., Moon J.D, & Jaffrey S.R. (2017). Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nature chemical biology, 13(11), 1187-1194.

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Purification of CD3ε-specific Antibody

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New Zealand White rabbit were immunized with the purified recombinant CD3ε according to the standard method. Rabbits were bled by cardiac puncture under deep terminal anesthesia and the serum were purified by protein G sepharose (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s protocols and guidelines. The antibody was further purified by an affinity column, which was prepared by coupling of recombinant CD3e–ΔTM protein to NHS-activated Sepharose (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions.

Miyazawa R., Murata N., Matsuura Y., Shibasaki Y., Yabu T, & Nakanishi T. (2018). Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp. Frontiers in Immunology, 9, 1321.

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Immobilization of Fluorescent Probes

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DFHBI and DFHO affinity matrix were prepared as described before^{20 (link),23 (link)}. Amine-functionalized DFHBI or DFHO (50 mM in DMSO) was diluted to 5 mM in 100 mM HEPES-KOH buffer (pH 7.5). 2 mL of the fluorophore solution was then added to 1 mL of NHS-activated Sepharose (GE Life Sciences) beads, which had been washed with 2 × 1 mL of cold reaction buffer. The beads were then incubated with amine-functionalized fluorophore solution overnight at 4^oC in the dark with gentle agitation. Then the beads were washed with the reaction buffer to remove the unreacted DFHBI or DFHO and incubated with 5 mL of 100 mM Tris.HCl (pH 8.0) for 2 h at room temperature to inactivate any remaining NHS-activated sites. After thorough washing with the reaction buffer and then with water, the beads were stored in 1:1 ethanol:0.1 M sodium acetate (pH 5.2) at 4^˚C. The efficiency of the coupling was calculated by quantifying the amount of free DFHBI or DFHO in the flow-through using absorbance. Using this approach, we estimated that the Sepharose beads contain approximately 5 μmol of DFHBI or DFHO per mL of resin after the coupling reaction.

Dey S.K., Filonov G.S., Olarerin-George A.O., Jackson B.T., Finley L.W, & Jaffrey S.R. (2021). Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag. Nature chemical biology, 18(2), 180-190.

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