Strep tactin xt

Manufactured by IBA Lifesciences

Sourced in Germany

Strep-Tactin® XT is a high-affinity streptavidin variant developed for the purification and detection of Strep-tagged proteins. It is designed to provide a robust and reliable system for the isolation and analysis of Strep-tagged biomolecules.

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Lab products found in correlation

Dna smart chip seq kit, by Takara Bio (2 mentions) Hiseq 4000, by Illumina (2 mentions) Truchip chromatin shearing kit, by Covaris (2 mentions) S220 focused ultrasonicator, by Covaris (2 mentions) Kapa hyperplus kit, by Roche (2 mentions) Biacore t200, by Cytiva (2 mentions) Nanodrop one, by Thermo Fisher Scientific (2 mentions) Cm5 sensor chip, by Cytiva (2 mentions) Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions) Spectrostar nano plate reader, by BMG Labtech (1 mentions) Twin strep tag capture kit, by IBA Lifesciences (1 mentions) Akta fplc, by GE Healthcare (1 mentions) Expi293f, by Thermo Fisher Scientific (1 mentions) Ni nta, by GenScript (1 mentions) Superdex 200 increase 10 300, by GE Healthcare (1 mentions) Pei max, by Polysciences (1 mentions) Biotin capture kit, by Cytiva (1 mentions) Hbs ep ph 7, by Cytiva (1 mentions) Evaluation software, by Cytiva (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions)

19 protocols using strep tactin xt

Strep-Tactin XT and H5-Strep-tag Oligomerization

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Strep-Tactin® XT (IBA Lifesciences, Goettingen, Germany) was mixed with H5-Strep-tag, resulting in final concentrations of 0, 1.6, 16, 160, 640 nM, 1.6, and 16 μM for Strep-Tactin® XT and 312.5 nm for H5-Strep-tag. Mixtures were rotated at room temperature for 30 min and used for the haemagglutination assay.
Oligomers formed by Strep-Tactin® XT and H5-Strep-tag at final concentrations of 160 nM and 312.5 nM, respectively, were designated H5 oligomers. These H5 oligomers were kept at 4°C to monitor their stability at different time points (1, 2, 6, and 36 days) by haemagglutination assay and to use for mouse immunization.

Phan H.T., Gresch U, & Conrad U. (2018). In vitro-Formulated Oligomers of Strep-Tagged Avian Influenza Haemagglutinin Produced in Plants Cause Neutralizing Immune Responses. Frontiers in Bioengineering and Biotechnology, 6, 115.

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E2 Protein Purification from HEK-293F Cells

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E2 proteins were purified from transfected supernatants of HEK-293F cells by affinity chromatography using Strep-Tactin^® XT (IBA Life Sciences, Göttingen, Germany) columns and following manufacturer’s recommendations. Briefly, supernatants were filtered through 0.2 µm filters and passed through the column at an approximate speed of 0.5 mL/min overnight. After two washing steps, bound E2 protein was eluted and concentrated in a Vivaspin 6 10 kDa cut-off filter (Sartorius, Göttingen, Germany) and left in the final buffer 20 mM Tris-HCl pH 8, 150 mM NaCl. Next, proteins were further fractionated by size-exclusion chromatography (SEC). The protein concentration was determined using Nanodrop with the theoretical extinction coefficient calculated via Expasy (ProtParam tool): E2 = 84.5 and E2.C8A = 85.

Marín M.Q., Sliepen K., García-Arriaza J., Koekkoek S.M., Pérez P., Sorzano C.Ó., Gómez C.E., Sanders R.W, & Esteban M. (2020). Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV. Vaccines, 8(3), 440.

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Monoclonal Antibody ELISA Binding Assay

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Two different ELISAs were performed to check the binding of the monoclonal antibodies and plasma to the recombinant HA proteins; pre-coated Ni-NTA (HisSorb, Qiagen) or Strep-Tactin XT (iba Lifesciences GmbH, Gӧttingen, Germany) ELISA plates. The recombinant proteins were coated at a 1 µg/mL final concentration for 2 h, followed by a washing step with TBS and residual blocking of 30–60 min with 2% skimmed milk in TBS (w/v). After a TBS wash, the primary antibody (starting at 3 µg/mL antibody or 1:100 diluted plasma) was three-fold serially diluted and incubated for 2 h, followed by a TBS wash. The secondary antibody labelled with horseradish peroxidase (HRP) was diluted 1:3000 or 1:10,000 (goat-anti-mouse in 0.1 mg/mL or goat-anti-human in 1 mg/mL respectively (KPL antibodies & conjugates)) and incubated for 1 h. Next, a washing step was performed with TBS/Tween-0.05%, the substrate supplemented with 1% TMB and 0.01% hydrogen peroxide was added, and the color development was stopped with 0.8 M sulfuric acid. The readout was done using a SPECTROstar Nano plate reader (BMG Labtech, De Meern, The Netherlands) and the OD was measured at 450 nm. All ELISAs were performed in duplicate, on two independent days (n = 4).

Aartse A., Eggink D., Claireaux M., van Leeuwen S., Mooij P., Bogers W.M., Sanders R.W., Koopman G, & van Gils M.J. (2021). Influenza A Virus Hemagglutinin Trimer, Head and Stem Proteins Identify and Quantify Different Hemagglutinin-Specific B Cell Subsets in Humans. Vaccines, 9(7), 717.

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Genome-Wide Mapping of RORα and RORγt

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RORα-TS and RORγt ChIP-Seq was performed as described (Ciofani et al., 2012 (link)) with the following modifications. For each ChIP, 20–80 million cells were cross-linked with paraformaldehyde; chromatin was isolated using truChIP Chromatin Shearing Kit (Covaris) and fragmented with a S220 Focused-ultrasonicator (Covaris). Twin-strep (TS) tagged RORα protein was precipitated using Strep-TactinXT according to the manufacturer’s protocol (IBA Lifesciences). Following immunoprecipitation, the protein-DNA crosslinks were reversed and DNA was purified. DNA from control samples was prepared similarly but without immunoprecipitation. Sequencing libraries were made from the resulting DNA fragments for both ChIP and controls using DNA SMART^™ ChIP-Seq Kit (Takara) for RORα-TS ChIP-Seq and KAPA HyperPlus Kit (Roche) for RORγt ChIP-Seq. The ChIP-Seq libraries were sequenced with paired-end 50 bp reads on an Illumina HiSeq 4000.

Hall J.A., Pokrovskii M., Kroehling L., Kim B.R., Kim S.Y., Wu L., Lee J.Y, & Littman D.R. (2022). Transcription factor RORα enforces stability of the Th17 cell effector program by binding to a Rorc cis-regulatory element. Immunity, 55(11), 2027-2043.e9.

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KRAS Variant Binding Kinetics

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Samples were thawed on ice, centrifuged at 13,000g for 10 min, transferred to a new tube, and quantified using a NanoDrop One (ThermoFisher). Binding kinetics and affinity of KRAS variants for RAS or K55 were evaluated by surface plasmon resonance on a BIAcore T200 instrument (Cytiva) with SPR running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.2). The assay format involved a Series S CM5 chip functionalized with Streptactin (50 μg ml⁻¹). In brief, amine coupling was used to create a Streptactin surface (Strep-Tactin XT) following instructions provided with the Twin-Strep-tag capture kit (IBA Lifesciences). Twin-Strep-tagged RAS or K55 protein constructs were captured on flow cell 4, leaving flow cell 3 as a subtractive reference. Capture levels of RAS or K55 were targeted between 50 and 100 resonance units, after which increasingly concentrated samples of KRAS variants were flowed over immobilized RAS or K55 (50 μl min⁻¹ for 1 min) and allowed to dissociate up to 3 min. A concentration series of each KRAS variant ranging from 0.74 nM to 60 nM was used to analyse binding to RAS or K55. The capture surface was regenerated with a 60 s injection of 3 M guanidine hydrochloride (50 μl min⁻¹ for 1 min). All sensograms were analysed using a 1:1 Langmuir binding model.

Weng C., Faure A.J., Escobedo A, & Lehner B. (2023). The energetic and allosteric landscape for KRAS inhibition. Nature, 626(7999), 643-652.

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Purification of TdfH-CP Complex

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For large-scale complex formation, refolded TdfH in 1x PBS, pH 7.4, 0.02% LMNG was applied and the flowthrough reapplied to a 1 mL StrepTactinXT (IBA Lifesciences) column attached to an Akta FPLC automated purification system (GE Healthcare). The column was washed and then CP (preincubated with CaCl₂ and ZnCl₂) was applied and the flowthrough reapplied 2x, the column was washed again, and then the complex was eluted using sample buffer supplemented with 3 mM desthiobiotin and the fractions analyzed by SDS-PAGE. Fractions containing the TdfH-CP complex were then pooled and concentrated to ~4 mg/mL. One limiting factor of this study is our inability to fully saturate CP with zinc, something that became obvious during our cryoEM studies. CP has a high propensity to precipitate out of solution before even reaching a 1:1 (zinc:CP) ratio; a ratio of 2:1 would be needed to fully saturate CP. From analysis of our cryoEM results, this leads to heterogeneity of the complex and limits the number of zinc saturated TdfH-CP particles that can be analyzed.

Bera A.K., Wu R., Harrison S., Cornelissen C.N., Chazin W.J, & Noinaj N. (2022). TdfH selectively binds metal-loaded tetrameric calprotectin for zinc import. Communications Biology, 5, 103.

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Purification of SARS-CoV-2 RBD Proteins

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The recombinant proteins were overexpressed in E. coli BL21 (DE3). After overnight induction by 0.1 mM isopropyl β-D-thiogalactoside (IPTG) at 16 °C in LB medium, cells were harvested and suspended in buffer (40 mM Tris-HCl, pH 8.0; 500 mM NaCl; 2mM phenylmethylsulfonyl fluoride). After cell lysis and centrifugation, the recombinant proteins were purified by Ni-NTA (GenScript) affinity chromatography. Ion exchange columns and gel-filtration chromatography (SD200, Superdex200 Increase 10/300, GE Healthcare) were used for further purification.
SARS-CoV-2 RBD wild-type proteins (with an N-terminal signal peptide and a C-terminal TwinStrep tag or 8 × His tag or TwinStrep-KKETPV tag) were expressed in Expi293F (Thermo Fisher Scientific) cells at 37 °C in a humidified 5% CO₂ incubator rotating at 120 r.p.m. Transfections were performed using PEI MAX (Polysciences) at a DNA:PEI ratio of 1:3. After cultivation for 96 h the supernatants were collected and purified by Ni-NTA (GenScript) affinity chromatography or Strep-Tactin® XT (IBA Lifesciences) according to the manufacturer's protocol. Ion exchange columns and gel-filtration chromatography (SD200, Superdex200 Increase 10/300, GE Healthcare) were used for further purification. Recombinant hACE2 protein was produced as described previously with some modifications [22] (link).

Pei G., Xu W., Lan J., Wang X, & Li P. (2022). CEBIT screening for inhibitors of the interaction between SARS-CoV-2 spike and ACE2. Fundamental Research, 2(4), 562-569.

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Measuring Receptor Binding Kinetics

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Measurements were performed using a Biacore T200 instrument, in triplicate for monomeric human and mouse ACE2 and duplicate for dimeric mouse ACE2. A CM5 chip covalently immobilized with StrepTactin XT (IBA LifeSciences) was used for surface capture of TwinStrepTag-containing RBDs (Wuhan-Hu-1, Alpha, Beta, Omicron, K417N) and a Cytiva Biotin CAPture Kit was used for surface capture of biotinylated RBDs (Delta and Wuhan-Hu-1 used for fold-change comparison to Delta). Two different batches of Omicron RBD were used for the experiments. Running buffer was HBS-EP+ pH 7.4 (Cytiva) and measurements were performed at 25 °C. Experiments were performed with a 3-fold dilution series of human ACE2 (300, 100, 33, 11 nM) or mouse ACE2 (900, 300, 100, 33 nM) and were run as single-cycle kinetics. Monomeric ACE2 binding data were double reference-subtracted and fit to a 1:1 binding model using Biacore Evaluation software. High concentrations of dimeric mouse ACE2 exhibited significant binding to the CAP sensor chip reference flow cell.

Cameroni E., Bowen J.E., Rosen L.E., Saliba C., Zepeda S.K., Culap K., Pinto D., VanBlargan L.A., De Marco A., di Iulio J., Zatta F., Kaiser H., Noack J., Farhat N., Czudnochowski N., Havenar-Daughton C., Sprouse K.R., Dillen J.R., Powell A.E., Chen A., Maher C., Yin L., Sun D., Soriaga L., Bassi J., Silacci-Fregni C., Gustafsson C., Franko N.M., Logue J., Iqbal N.T., Mazzitelli I., Geffner J., Grifantini R., Chu H., Gori A., Riva A., Giannini O., Ceschi A., Ferrari P., Cippà P.E., Franzetti-Pellanda A., Garzoni C., Halfmann P.J., Kawaoka Y., Hebner C., Purcell L.A., Piccoli L., Pizzuto M.S., Walls A.C., Diamond M.S., Telenti A., Virgin H.W., Lanzavecchia A., Snell G., Veesler D, & Corti D. (2021). Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. Nature, 602(7898), 664-670.

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Affinity Purification of Strep-Tagged Proteins

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Cell pellets from infected suspension cultures (1 mL for 25 × 10⁶ cells) were disrupted by sonication in buffer A (20 mM Tris/HCl pH8, 250 mM NaCl, 0.1% NP40, 1 mM DTT and EDTA free protease inhibitor cocktail (Roche)) and the clarified lysate subjected to Strep-Tactin^®XT affinity chromatography (IBA-Lifesciences). Proteins were eluted in the same buffer supplemented with 10 mM desthiobiotin.

Kolesnikova O., Zachayus A., Pichard S., Osz J., Rochel N., Rossolillo P., Kolb-Cheynel I., Troffer-Charlier N., Compe E., Bensaude O., Berger I, & Poterszman A. (2022). HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system. Scientific Reports, 12, 2030.

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SPR Analysis of NMDAR-Neuroligin 1 Binding

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SPR experiments were performed on a Biacore T200 machine (GE Healthcare) operated at a data collection frequency of 10 Hz; i.e. a temporal resolution of 0.1 s. Streptactin XT (IBA Lifesciences) was chemically coupled via amine coupling chemistry onto CM5 chips to a response unit (RU) level of 5000 RU. Then, OneStrep-tagged rat GluN1a-GluN2B heterotetrameric NMDA receptor (NMDAR) was captured to a level of 5000 RU. SPR running buffer composition was 200 mM NaCl, 20 mM HEPES pH 7.4, 10 mM Glycine, 10 mM Glutamate, 3mM CaCl₂, 0.010% LMNG. For the interaction with NL1(–A–B)_ECTO, a single-cycle kinetics (SCK) approach was adopted. Injection of 5 concentrations of NL1(–A–B)_ECTO, prepared in a two-fold dilution series from a 25 μM stock concentration, was performed in order of increasing concentration. Each sample was injected for 120 s at a flow rate of 25 μL/min, followed by a 60 s intermittent dissociation phase or a final 600 s dissociation phase.

Elegheert J., Cvetkovska V., Clayton A.J., Heroven C., Vennekens K.M., Smukowski S.N., Regan M.C., Jia W., Smith A.C., Furukawa H., Savas J.N., de Wit J., Begbie J., Craig A.M, & Aricescu A.R. (2017). Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins. Neuron, 95(4), 896-913.e10.

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