Zeba spin column

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Zeba spin columns are a fast and efficient way to desalt and buffer exchange small volume samples. They utilize a proprietary resin to rapidly remove salts, buffers, and other small molecules from protein, peptide, or nucleic acid solutions.

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Zeba desalt spin columns (48 citations) Zeba columns (44 citations) Spin columns (29 citations) Zeba Micro Spin Desalting Columns (17 citations) Zeba Spin 7K MWCO desalting columns (12 citations) Zeba Spin Desalting Columns 7K MWCO 0.5 mL (10 citations) Zeba spin desalt columns (3 citations) Zeba spin columns (7K MWCO; (3 citations) Zeba Spin Desalting Columns 40K (3 citations) Zeba™ desalting spin columns 7 K MWCO (3 citations)

105 protocols using zeba spin column

Protein Labeling and Purification Protocol

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Purified syt1, cpx2, and EIIA^Glc were desalted using Zeba Spin columns (Thermo Fisher) in buffer D (50 mM Tris-HCl, pH 8, 100 mM NaCl, 5% glycerol) and labeled with a 3-fold excess of IANBD amide or OG maleimide in the presence of TCEP (0.2 mM) at room temp for 2 h. Free dyes were removed by passing through Zeba Spin columns in buffer A.
For fluorescence fluctuation spectroscopy experiments, spMSP1D1, spNW30, and spNW50 were desalted using Zeba Spin columns in PBS buffer and labeled with a 3-fold excess of fluorescein isothiocyanate (FITC) at room temp for 2 h. Free dyes were removed by passing through Zeba Spin columns in buffer A.

Zhang S., Ren Q., Novick S.J., Strutzenberg T.S., Griffin P.R, & Bao H. (2021). One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 12, 5451.

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Assessing Binding Interactions of mCRP with Nicotine, Acetylcholine, and Tacrine

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Surface plasmon resonance (SPR) was used to assess the binding of mCRP to nicotine, acetylcholine, tacrine and 3H12 polyclonal antibody. Monomeric CRP was first buffer exchanged using a 0.5 ml Zeba^TM spin column (Thermo) which was pre-equilibrated in PBS. mCRP was then incubated with a 1:2 ratio of NHS-Peg4-Biotin (Thermo) for 30 min at room temperature before purifying the free biotin with another Zeba^TM spin column in PBS. SPR was performed on a Biacore T200 (GE life sciences) and a streptavidin coated SA chip. The instrument was equilibrated in Biacore buffer, 10 mM HEPES buffer pH 7.4 with 0.05% tween 20 and the SA chip was washed with 10 mM EDTA and 50 mM NaOH prior to loading with biotinylated mCRP. For the antibody binding test the chip was loaded with ~100 response units of mCRP. For the small molecule tests the chip was loaded to a maximum loading of ~3,500 response units of biotinylated mCRP. Analytes, either antibody at 1:100 dilution, 1 mM nicotine, 1 mM acetylcholine, or 10 μM tacrine diluted in Biacore buffer were injected at 30 μl/min over the mCRP surface and the response monitored in real time. The results are shown as a subtracted response where flow cell 1 was used as a reference with no mCRP added which was subsequently subtracted from the data.

Slevin M., Iemma R.S., Zeinolabediny Y., Liu D., Ferris G.R., Caprio V., Phillips N., Di Napoli M., Guo B., Zeng X., AlBaradie R., Binsaleh N.K., McDowell G, & Fang W.H. (2018). Acetylcholine Inhibits Monomeric C-Reactive Protein Induced Inflammation, Endothelial Cell Adhesion, and Platelet Aggregation; A Potential Therapeutic?. Frontiers in Immunology, 9, 2124.

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LPS Monomeric Labeling and Purification

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LPS (10 mg, O55:B5 E. coli, Sigma-Aldrich) was made monomeric by treatment with 5 ml of 0.5% triethylamine (Sigma-Aldrich) and by sonication for 15 min on ice. After the sonication, 200 μl of LPS was removed from the solution and added to a tube containing NaIO4 (20ul,20 mM, made freshly), pH 7.1. Excess NaIO4 was removed on a Zeba spin column (Thermo Scientific, IL.) after incubated 30 min on ice, reacted with 26ul of DOTH (7.8 mM in H2O, 202 nmol), pH 6.2, at RT for 2 h., and then treated with 10ul of sodium cyanoborohydride (200 mM in H2O, 2000 nmol) at RT for 2 h., followed by running a Zeba spin column again to remove excess DOTA and NaCNBH3. All reaction was protected from light [73 (link)]. Preparation of FAM-LPS follow the protocol of the FAM conjugation from company.

Zhang Z., La Placa D., Nguyen T., Kujawski M., Le K., Li L, & Shively J.E. (2019). CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes. BMC Immunology, 20, 7.

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Protein Labeling with 5-IAF Fluorophore

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We labeled protein of interests using 5-IAF to allow quantification via in-gel fluorescence. Purified proteins were desalted using Zeba Spin columns (Thermo Fisher) in buffer D (50 mM Tris-HCl, pH 8, 100 mM NaCl, 5% glycerol) and labeled with a 3-fold excess of 5-IAF in the presence of TCEP (0.2 mM) at room temp for 3 h. We removed excessed dyes using Zeba Spin columns in buffer A.

, & Bao H. (2021). Developing Nanodisc-ID for label-free characterizations of membrane proteins. Communications Biology, 4, 514.

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Antibody-Drug Conjugate Characterization

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Bioconjugation, purification, and HPLC analysis were performed as previously described [35 (link)]. Aldehyde-tagged antibodies (15 mg/mL) were conjugated to HIPS-linker-payloads (8 mol equiv DAR) for 72 h at 37 °C in 20 mM sodium citrate, 50 mM NaCl pH 5.5 and up to 2.5% DMA. Free drug was removed using a 40 kDa Zeba^TM spin column (Thermo Fisher Scientific, Waltham, MA, USA) equilibrated with 20 mM sodium citrate, 50 mM NaCl pH 5.5. To determine the DAR of the final product, ADCs were examined by analytical HIC (Cat. #14947 Tosoh Corp., Tokyo, Japan) with mobile phase A: 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0, and mobile phase B: 25% isopropanol, 18.75 mM sodium phosphate pH 7.0. To determine aggregation, samples were analyzed using analytical size exclusion chromatography (SEC; Cat. #08541, Tosoh Corp., Tokyo, Japan) with a mobile phase of 300 mM NaCl, 25 mM sodium phosphate pH 6.8.

Harel E.T., Drake P.M., Barfield R.M., Lui I., Farr-Jones S., Van’t Veer L., Gartner Z.J., Green E.M., Lourenço A.L., Cheng Y., Hann B.C., Rabuka D, & Craik C.S. (2019). Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer. Antibodies, 8(4), 54.

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Nsp1 C-K Protein Labeling Protocol

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Nsp1 C-K (3 mg/mL) was desalted using a 7K MWCO, 0.5 mL ZebaTM spin column (ThermoFisher) into a buffer containing 20mM HEPES-KOH pH 7.5, 120mM KOAc, 5mM Mg(OAc)₂. A 1:5 mole ratio of protein to TAMRA maleimide, 6-isomer (lumiprobe Life science solutions) was used for each reaction. The reaction was gently mixed and placed at RT for 1 hr protected from light. The protein solution was then desalted once more to remove any free probe into a buffer containing 20mM HEPES-KOH pH 7.5, 120mM KOAc, 5mM Mg(OAc)₂, 5% glycerol and 1mM TCEP. The dye:protein ratio for each labeled protein was between 0.84-0.91. Final protein concentration was determined by A280 and generally yielded a ∼90 percent recovery.

Mendez A.S., Ly M., González-Sánchez A.M., Hartenian E., Ingolia N.T., Cate J.H, & Glaunsinger B.A. (2021). The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression. Cell Reports, 37(3), 109841.

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Conjugation of FGF1V to MMAE

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FGF1_V solution (30 µM) in 25 mM phosphate buffer, pH 7.4, and 100 mM NaCl was reduced with 1 mM TCEP for 20 minutes at room temperature, desalted with a Zeba spin column (Thermo Fisher Scientific, Waltham, MA, USA), and added to a CH₃CN solution of linker-functionalized MMAE (vcMMAE) containing a maleimide moiety, and the conjugation was carried out at 4°C. There was a two- to fivefold molar excess of the drug over the FGF1_V N-terminal –SH group. The reaction was quenched after 16 hours with an excess of free cysteine. Different reaction conditions and durations were tested in order to achieve optimum conjugation efficiency with protein structure and function retained. Reaction progress was monitored by SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
To purify the conjugate, unmodified FGF1_V was removed by hydrophobic interaction chromatography on phenyl-Sepharose (GE Healthcare, Chicago, IL, USA). The conjugation reaction mixture was loaded on a phenyl-Sepharose column equilibrated in 25 mM Tris-HCl, pH 7.4, and 2 M NaCl, and FGF1_V–vcMMAE was eluted with a linear gradient of decreasing salt concentration (from 0% to 100% of 25 mM Tris-HCl, pH 7.4, 0.1 M NaCl). Identity and purity of conjugated FGF1_V–vcMMAE were confirmed by SDS-PAGE, Western blotting, and MALDI-TOF MS.

Szlachcic A., Zakrzewska M., Lobocki M., Jakimowicz P, & Otlewski J. (2016). Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy. Drug Design, Development and Therapy, 10, 2547-2560.

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Photocrosslinking of Di-/Tri-Ub by MTS-alkynyldiazirine

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A photocrosslinker was installed on different subunits of K48 di-Ub and K6/K48 tri-Ub using a Ub variant in which A46 is replaced with Cys (A46C). The A46C bearing Ub chains were incubated with 500 µM MTS-alkynyldiazirine (Redbrick molecular) in 25 mM Tris, pH 8, 10% DMSO at room temperature for 30 min. Excess photocrosslinker was removed with Zeba spin column (Thermo Fisher scientific). The modified Ub chains (15 μM) were incubated with UCH37^C88A•RPN13^DEUBAD (45 µM) in crosslink reaction buffer (50 mM HEPES, pH 7.4, 50 mM NaCl) on ice for 1 hr. The mixture was then irradiated with a UV lamp (Omnicure, 320–500 nm) for 5 min on ice, followed by separation with SDS-PAGE and Coomassie staining. The gel slice containing crosslinked UCH37-Ub chain conjugate was cut out and reduced with 10 mM DTT (Ub peptide is released from the crosslinked peptide, but UCH37 peptide is still modified with the crosslinker) and then alkylated with 55 mM iodoacetamide. In-gel digestion was done with trypsin at 37°C overnight.

Du J., Babik S., Li Y., Deol K.K., Eyles S.J., Fejzo J., Tonelli M, & Strieter E. (2022). A cryptic K48 ubiquitin chain binding site on UCH37 is required for its role in proteasomal degradation. eLife, 11, e76100.

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Protein Ionic Strength Optimization

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Protein samples were prepared at 10 μM concentration in phosphate buffer (20 mM phosphate, pH 7.6), using NaCl to vary ionic strength (40, 90, 140, 190, 240, 340, 440, 540, 640 mM). To ensure accurate ionic strengths, samples were buffer exchanged on a Zebaspin column (ThermoFisher Scientific) equilibrated with the appropriate buffer. Samples were incubated overnight at 4 °C to allow for equilibration.

Ping J., Pulsipher K.W., Vishnubhotla R., Villegas J.A., Hicks T.L., Honig S., Saven J.G., Dmochowski I.J, & Johnson A.T. (2017). Structural-functional analysis of engineered protein-nanoparticle assemblies using graphene microelectrodes †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc01565h Click here for additional data file.. Chemical Science, 8(8), 5329-5334.

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APAP-CYS Quantification in Liver

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The APAP-CYS content in liver was measured by LC-MS/MS following the previously described method^{71 (link)}. Liver protein extract was filtered through a desalting column (Zeba spin column, Thermo Fisher) pre-equilibrated with 50 mM (NH₄)₂HCO₃ following the manufacturer’s instructions. An aliquot (45 µl) of the filtrate was mixed with 5 µl of protease type XIV (80 U/ml) (Sigma) and incubated for 24 hours at 37 °C to liberate APAP-CYS. After digestion, 5 µL of norbuprenorphine-d3 (100 µg/ml, Cerilliant Corp.) was added as an internal standard, followed by acetonitrile (300 µL) for precipitation. Dry pellet was obtained by evaporation at 13 psi, and was reconstituted in 100 µL of 2% acetonitrile buffer with 0.1% formic acid. The reconstituted solution was cleared by centrifugation and the supernatant were transferred to a clean vial for LC-MS/MS (Thermo TSQ Quantiva triple-quadrupole mass spectrometer coupled with a Shimadzu Nexera UHPLC). Free APAP-CYS (m.w. 270, Cayman #26388) was used for the quantification standard.

Lee H., Lee T.J., Galloway C.A., Zhi W., Xiao W., de Mesy Bentley K.L., Sharma A., Teng Y., Sesaki H, & Yoon Y. (2023). The mitochondrial fusion protein OPA1 is dispensable in the liver and its absence induces mitohormesis to protect liver from drug-induced injury. Nature Communications, 14, 6721.

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