Vivaspin device

Manufactured by Sartorius

Sourced in Germany

Vivaspin devices are centrifugal concentrators used for the separation and concentration of macromolecules such as proteins, nucleic acids, and other biological samples. They feature a membrane-based filtration system that allows for the selective retention of target molecules while allowing smaller molecules to pass through.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions) Sinapinic acid matrix, by Merck Group (1 mentions) Autoflex mass spectrometer, by Bruker (1 mentions) Flexanalysis software, by Bruker (1 mentions) Hiprep 26 10 desalting column, by GE Healthcare (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Hitrap heparin column, by GE Healthcare (1 mentions) Total exosome isolation reagent, by Thermo Fisher Scientific (1 mentions) 13c d glucose, by Cambridge Isotopes (1 mentions) 15n ammonium chloride, by Cambridge Isotopes (1 mentions) Expifectamine, by Thermo Fisher Scientific (1 mentions) Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) Titan krios electron microscope, by Thermo Fisher Scientific (1 mentions)

9 protocols using vivaspin device

MALDI-TOF Mass Spectrometry Analysis

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MALDI-TOF mass spectra were measured with an Autoflex mass spectrometer (Bruker Daltonics) operated on linear positive ion mode. External mass calibration of the instrument, for the m/z range of interest, was carried out using the monomeric (66.4 kDa) and dimeric (132.8 kDa) molecular ions of BSA (reference 7030; Sigma-Aldrich) as calibrants. Before MS analysis the protein samples (concentration around 10 μM; before and after cross-linking) were submitted to buffer exchange against 20 mM Tris (pH 7.5), 50 mM NaCl, using Vivaspin devices (Sartorius, cut-off of 30 kDa for Impβ alone and 10 kDa for Impβ/Rev). The buffer-exchanged protein samples were then mixed in a 1:2, 1:5 or 1:10 (vol/vol) ratio with sinapinic acid matrix (Sigma-Aldrich; 10 mg/ml in water/acetonitrile/TFA, 50/50/0.1, vol/vol/v) and 1–2 μl were deposited on the target and allowed to air dry at RT. Mass spectra data were processed with flexAnalysis software (v.3.0; Bruker Daltonics).

Spittler D., Indorato R.L., Boeri Erba E., Delaforge E., Signor L., Harris S.J., Garcia-Saez I., Palencia A., Gabel F., Blackledge M., Noirclerc-Savoye M, & Petosa C. (2022). Binding stoichiometry and structural model of the HIV-1 Rev/importin β complex. Life Science Alliance, 5(10), e202201431.

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Antibody-Drug Conjugate Production Protocol

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The antibody in SIP or IgG format was reduced with 30 equivalents
(calculated on the basis of antibody monomers, each containing a single cysteine
residue) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl, ACBR) in
phosphate buffer saline (PBS, p.H. = 7.4). The reduced protein was purified by
size exclusion chromatography on an HiPrep 26/10 Desalting column (GE
Healthcare); 1mM DTPA (Sigma-Aldrich)/PBS was used as a mobile phase at a flow
rate of 1.5-2 mL/min. The recovered protein was pooled and concentrated using
Vivaspin^® Turbo 15 (Sartorious) in order to remain in the
capacity limit of the FPLC-loop. Ten equivalents of Vedotin (MC-vc-PAB-MMAE,
Concortis Biosystems) were dissolved in DMSO (Sigma-Aldrich) and added to the
reduced protein with a final DMSO content of 5% (v/v). IgG and Vedotin were let
react under stirring for 15’ at RT, the reaction was then quenched with
L-Cys (Fluka) at a final concentration of 1 mM for 10’ at RT. Final
product was FPLC-purified as described previously. The ADCs were then formulated
at the desired concentration by centrifugation using Vivaspin devices
(Sartorious), snap-frozen in liquid nitrogen and stored at -80 °C until
further use.

Gébleux R., Stringhini M., Casanova R., Soltermann A, & Neri D. (2016). Non-internalizing antibody-drug conjugates display potent anti-cancer activity upon proteolytic release of monomethyl auristatin E in the sub-endothelial extracellular matrix. International journal of cancer, 140(7), 1670-1679.

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Recombinant SARS-CoV-2 and HCoV-OC43 Nucleoproteins

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Full-length SARS-CoV-2 and HCoV-OC43 nucleoproteins were produced by expression in Escherichia coli C43(DE3) cells (Lucigen) and purified as previously described.¹² (link) The SARS-CoV-2 nucleoprotein was produced with an N-terminal His₆ tag from pOPTH-1124 plasmid (kindly provided by Jakub Luptak and Leo James, Laboratory for Molecular Biology, Cambridge, UK). The HCoV-OC43 nucleoprotein (NCBI Reference Sequence: AOL02457.1 from isolate HCoV-OC43/human/Mex/LRTI_238/2011) was produced with an N-terminal SUMO-His₆ tag, which was removed during purification, by incubation with ubiquitin-like-specific protease-1 (ULP1) at 4°C overnight. Following incubation, the protein was diluted to 300 mM NaCl in 20 mM HEPES pH 8.0, loaded onto a 5 mL HiTrap heparin column (GE healthcare) and eluted over a linear 0.3-1M NaCl gradient. Purified nucleoproteins were concentrated by ultrafiltration using appropriate VivaSpin devices (Sartorius), were snap-frozen in liquid nitrogen in small aliquots and stored at −80°C.

Deakin C.T., Cornish G.H., Ng K.W., Faulkner N., Bolland W., Hope J., Rosa A., Harvey R., Hussain S., Earl C., Jebson B.R., Wilkinson M.G., Marshall L.R., O’Brien K., Rosser E.C., Radziszewska A., Peckham H., Patel H., Heaney J., Rickman H., Paraskevopoulou S., Houlihan C.F., Spyer M.J., Gamblin S.J., McCauley J., Nastouli E., Levin M., Cherepanov P., Ciurtin C., Wedderburn L.R, & Kassiotis G. (2021). Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases. Med (New York, N.y.), 2(9), 1093-1109.e6.

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Isolating Extracellular Vesicles from Cancer Cells

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Supernatants (SN) from MDA-MB-231 CSC and DCC cell cultures, after 48 hours incubation in EV-depleted medium, were centrifuged at increasing speeds: 300g at 4°C for 10 minutes, 2000g, 10 minutes 4°C and 10 000g 20 minutes 4°C to eliminate potential cellular and subcellular debris. Clarified SNs were concentrated through centrifugation at 5000g, 15 minutes, 4°C, using 300 000 kDa VIVAspin devices (Sartorius, Gottingen, Germany). EVs were then precipitated from concentrated SN by the addition of "Total Exosome Isolation Reagent" (Invitrogen, Waltham, MA) according to manufacturer's instructions and EVs resuspended in PBS.

González-Callejo P., Gener P., Díaz-Riascos Z.V., Conti S., Cámara-Sánchez P., Riera R., Mancilla S., García-Gabilondo M., Peg V., Arango D., Rosell A., Labernadie A., Trepat X., Albertazzi L., Schwartz S J.r., Seras-Franzoso J, & Abasolo I. (2023). Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs. International journal of cancer, 152(10).

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Purification and Labeling of OutD Domains

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PreScission protease to 5 ml of Glutathione Sepharose in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0 at 4°C for 6 h. N0 and N0-N1-N2 domains of OutD (residues 1 to 85 and 1 to 258 of signal peptide-less OutD, respectively, Table S4) were produced in E. coli BL21(DE3) and purified by nickel-affinity chromatography as described previously (33) . For NMR spectroscopy, uniformly 15 N-and 13 C-labeled proteins were produced by growing cell cultures in M9 minimal medium that contained 1 g/l 15 N-ammonium chloride and 2 g/l 13 C-D-glucose (Cambridge Isotope Laboratories, Inc.) as the sources of nitrogen and carbon, respectively. Prior to crystallization trials and NMR spectroscopy, the purified proteins were run on Superdex S200 size-exclusion column and concentrated with Vivaspin devices (Sartorius).

Zhang S., Gu S., Rycroft P., Ruaudel F., Delolme F., Robert X., Ballut L., Pickersgill R.W., & Shevchik V.E. (2021). Scaffolding protein GspB/OutB facilitates assembly of the Dickeya dadantii type 2 secretion system by anchoring the outer membrane secretin pore to the inner membrane and to the peptidoglycan cell wall.

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Antibody Fab Fragment Purification

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IgG-enriched supernatant following caprylic acid precipitation was diafiltrated into water or saline using Vivaspin device (Sartorius, Germany) with a 100 kDa molecular weight cut-off (MWCO) polyethersulfone membrane. F(ab')₂ sample, as well as the commercial pepsin preparation employed for its preparation, were dialfiltrated into 20 mM MES buffer + 0.15 M NaCl, pH 5.0, on a membrane with a MWCO of 50 kDa. In each diafiltration step the buffer was exchanged by a factor of 8,000 ×.

Kurtović T., Lang Balija M., Brgles M., Sviben D., Tunjić M., Cajner H., Marchetti-Deschmann M., Allmaier G, & Halassy B. (2019). Refinement strategy for antivenom preparation of high yield and quality. PLoS Neglected Tropical Diseases, 13(6), e0007431.

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Purification of BaL gp120-CD4-FLAG Protein

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BaL gp120-CD4-FLAG plasmid (500 ng per ml of cells) was transfected into Expi293F cells at 2 × 10⁶ cells/ml using Expifectamine (Life Technologies). Transfection enhancers (Life Technologies) were added 20 hours post-transfection, and the culture was centrifuged for 20 min at 4,000 × g after 200 hours. Protease inhibitors (SIGMAFAST inhibitor cocktail, Sigma) dissolved in PBS were added to the supernatant. The supernatant was incubated with anti-FLAG M2 affinity gel (Sigma) for 4 hours at 4 °C. The resin was collected by centrifugation at 1,000 × g for 5 min, washed with PBS, and transferred to a gravity column for elution. BaL gp120-CD4-FLAG was eluted with 5 column volumes of 200 μg/ml DYKDDDDK peptide (ApexBio) in PBS. Eluted protein was concentrated using a Vivaspin device (MWCO 100 kD, Sartorius), and further purified on a Superose 6 increase 10 300 GL (GE Life Sciences) size exclusion chromatography column equilibrated with PBS. Peak fractions were pooled and concentrated, and aliquots were snap frozen in liquid nitrogen before storage at −80 °C.

Heredia J.D., Park J., Brubaker R.J., Szymanski S.K., Gill K.S, & Procko E. (2018). Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning. Journal of immunology (Baltimore, Md. : 1950), 200(11), 3825-3839.

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IgG Purification and Diafiltration

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IgG preparations obtained from all purification procedures were diafiltrated using Vivaspin device (Sartorius, Germany) with a 100 kDa molecular weight cut-off (MWCO) polyethersulfone membrane. Samples from ASP and CEX processing were first desalted by diafiltration into 50 mM MES buffer, pH 5.5. All final products, including those of lower purity that were submitted to additional caprylic acid treatment step, were diafiltrated into 0.2 M phosphate buffer, pH 6.0, ensuring matrix uniformity prior further analysis. In each diafiltration step the buffer exchange factor was approximately 8000.

Mateljak Lukačević S., Kurtović T., Lang Balija M., Brgles M., Steinberger S., Marchetti-Deschmann M, & Halassy B. (2020). Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches. Toxins, 12(12), 798.

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Cryo-EM Imaging of Csu Pili

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Supernatant containing detached Csu fimbriae was concentrated to approximately 10 g l⁻¹ using a Vivaspin device (Sartorius Stedim) with a molecular mass cut‐off of 100 kDa. Then, 4 µl of sample was applied to glow-discharged Quantifoil R2/2 300 mesh copper grids coated with ultrathin carbon (Electron Microscopy Sciences). The grids were blotted and plunge-frozen into liquid ethane using the Vitrobot Mark IV (Thermo Fisher Scientific) at 4 °C and 100% humidity. The data were collected on a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a Gatan K3 direct electron detector operated in super-resolution mode with a pixel size of 0.433 Å and a defocus range of −1.0 to −3.0 μm. A total dose of 60 electrons per Å was applied and equally divided among 40 frames to allow for dose weighting. SerialEM (v.3.6) was used for automated cryo-EM data collection. Details on cryo-EM data collection are summarized in Supplementary Table 1. A representative cryo-EM micrograph of Csu pili is shown in Supplementary Fig. 2.

Pakharukova N., Malmi H., Tuittila M., Dahlberg T., Ghosal D., Chang Y.W., Myint S.L., Paavilainen S., Knight S.D., Lamminmäki U., Uhlin B.E., Andersson M., Jensen G, & Zavialov A.V. (2022). Archaic chaperone–usher pili self-secrete into superelastic zigzag springs. Nature, 609(7926), 335-340.

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