Zeba desalt spin column

Manufactured by Thermo Fisher Scientific

Sourced in United States

Zeba desalt spin columns are laboratory equipment designed to remove salts, buffers, and other small molecules from protein samples. They work by using a size-exclusion resin to selectively retain the protein of interest while allowing smaller molecules to pass through. This process is known as buffer exchange or desalting.

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Lab products found in correlation

Sulfo nhs biotin, by Thermo Fisher Scientific (3 mentions) Hrp conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions) Surfact amps nonidet p 40, by Thermo Fisher Scientific (3 mentions) Gapdh, by Santa Cruz Biotechnology (3 mentions) Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions) Quant tag biotin kit, by Vector Laboratories (2 mentions) Bis nhs ester dye fluorophores, by GE Healthcare (2 mentions) Hitrap, by Cytiva (2 mentions) Anti pan akt, by Cell Signaling Technology (2 mentions) Anti pser473 akt, by Cell Signaling Technology (2 mentions) Streptavidin hrp, by GE Healthcare (2 mentions) Protease inhibitor mixture tablet, by Roche (2 mentions) Streptavidin chip, by GE Healthcare (2 mentions) Anti flag m2 bead, by Merck Group (2 mentions) Anti ptp1b fg6, by Merck Group (2 mentions) Pt 66 agarose conjugated beads, by Merck Group (2 mentions) Anti ptp1b pser50 ab62320, by Abcam (2 mentions) Ez link biotin iodoacetyl peg2 biotin iap, by Thermo Fisher Scientific (2 mentions) Biacore sensor nta, by GE Healthcare (2 mentions) Anti flag m2 peroxidase, by Merck Group (2 mentions)

Spelling variants of this product

Zeba spin columns (105 citations) Zeba columns (44 citations) Spin columns (29 citations) Zeba spin desalt columns (3 citations) Desalt columns (2 citations)

48 protocols using zeba desalt spin column

Biotinylation of Allergen Proteins

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EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, Rockford, IL) was added to a defined amount of each allergen (2 mg for chitinase, 0.25 mg for Bla g 3) at a 10-fold molar excess and incubated for 30 min at room temperature. The biotinylated mix was put over a pre-washed Zeba Desalt Spin Column (Thermo Scientific, Rockford, IL) two times and the concentration was determined after biotinylation by Advanced Protein Assay (Cytoskeleton, Denver, Colorado).
The quantification of biotinylation was carried out by using a Quant Tag™ Biotin Kit (Vector Laboratories, Burlingame, CA). Samples were tested in triplicate against a known biotin standard curve to determine the number of biotins per allergen molecule.

Pomés A., Schulten V., Glesner J., da Silva Antunes R., Sutherland A., Bacharier L.B., Beigelman A., Busse P., Frazier A, & Sette A. (2021). IgE and T Cell Reactivity to a Comprehensive Panel of Cockroach Allergens in Relation to Disease. Frontiers in Immunology, 11, 621700.

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Biotinylation and Functionalization of Antibodies

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Dynabeads My One Streptavidin T1 were selected. They are 1

μ

m diameter homogeneous polymer particles embedding superparamagnetic iron oxide nanoparticles. They have been functionalized with two different monoclonal antibodies of the same IgG2a isotype: a rat anti-CD138 mAb (BD Pharmingen) and a murine mAb, IpaD-315 (described in Section 2.4), according to the commercial protocol after their biotinylation and purification.
For mAb biotinylation, 100

μ

g of antibodies were diluted in 400

μ

L of 0.1 M borate buffer pH 9.0 containing 6

μ

L of biotin (Sigma-Aldrich) in DMF at 1 mg/mL and incubated for 30 min at room temperature. Then, 100

μ

L of 1 M Tris HCl buffer pH 8.0 were added and incubated for 15 min. Finally, the biotinylated mAb was purified from free biotin on Zeba Desalt Spin column (Thermo Scientific) in 0.1 M potassium phosphate buffer pH 7.4 with 0.15 M NaCl. The absorbance of the final solution was measured between 280 and 320 nm to determine the concentration of the purified biotinylated antibody. biotinylated antibodies were then mixed at room temperature with streptavidin coated beads for 30 min, washed four times in PBS 0.1% BSA and stored in PBS 0.1% BSA at 4

^{\circ}

C until use.

Giraud M., Delapierre F.D., Wijkhuisen A., Bonville P., Thévenin M., Cannies G., Plaisance M., Paul E., Ezan E., Simon S., Fermon C., Féraudet-Tarisse C, & Jasmin-Lebras G. (2019). Evaluation of In-Flow Magnetoresistive Chip Cell—Counter as a Diagnostic Tool. Biosensors, 9(3), 105.

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Fluorescent Antibody Conjugation Protocol

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Rabbit and mouse anti-human antibodies were first purified on HiTrap Protein A or Protein G columns respectively to remove additives and stabilizers. Buffer exchange and concentration was conducted using Amicon 30K filters and purified antibody was resuspended in PBS buffer at a final concentration of 1mg/mL. Direct conjugation to Cy2-, Cy3- and Cy5-Bis NHS Ester dye fluorophores (GE Healthcare PA12000, PA13000, PA15000) was done according to manufacturer’s instructions. The conjugation reaction was prepared with a minimum loading of 35ug of protein, 10% w/v of NaHCO₃ (pH 8.5), and dye conjugate and incubated for 60 minutes in the dark at room temperature. Following the conjugation, the reaction was loaded on a Zeba desalt spin column (ThermoFisher, Waltham, MA) to remove unbound fluorophore, and the final concentration and dye:protein ratio measured by Nanodrop. Following conjugation, the antibody solution was adjusted to a final concentration between 150–300 μg/mL and stabilized with 1% BSA and 0.45% sodium azide.

Maus R.L., Leontovich A.A., Moore R.M., Becher L., Nevala W.K., Flotte T.J., Guo R., Schimke J.M., Dicke B.A., Yan Y, & Markovic S.N. (2022). Resolving the Heterogeneous Tumor-Centric Cellular Neighborhood through Multiplexed, Spatial Paracrine Interactions in the Setting of Immune Checkpoint Blockade. Cancer Research Communications, 2(2), 78-89.

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CD Spectroscopy Analysis of Protein Redox State

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Circular dichroism spectra were obtained on a CD spectrophotometer (Applied Photophysics, UK) using a 10 mm cuvette equilibrated at 30°C. All the samples were desalted and the buffer exchanged into PBS (pH 8.0) through a Zeba™ Desalt Spin Column (Thermo Scientific). One millimolar H₂O₂ and 2.5 mM DTT as final concentrations were incubated in the corresponding samples during equilibration for 20 minutes. Sample concentrations were diluted to 0.15 μM. The CD spectra were collected between 190 nm and 280 nm, and 3 repeat scans were averaged. Parameters of secondary structure were calculated by the CD Spectra Deconvolution Program CDNN version 2.1.

Wei Z.H., Chen H., Zhang C, & Ye B.C. (2014). FRET-Based System for Probing Protein-Protein Interactions between σR and RsrA from Streptomyces Coelicolor in Response to the Redox Environment. PLoS ONE, 9(3), e92330.

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Fluorescent Diabody Conjugation Protocol

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To achieve optimal conjugation efficiencies, the diabody was first concentrated using an Amicon^® Ultra-0.5mL (10K) Centrifugal Filter Device (Millipore, Carrigtwohill, County Cork, Ireland) to a concentration greater than 2.8 mg/mL. Then, 50 μM diabody was reduced in 40-fold molar excess of TCEP for 2 hours at room temperature. A 20-fold molar excess of Cy5 Maleimide dissolved in dimethylformamide was then added to the reduced diabody and the mixture was incubated for 2 hours at room temperature. After incubation, excess dye was removed using a 2 mL Zeba Desalt Spin Column (Thermo Scientific). Cy5 and diabody concentrations were then measured using a spectrophotometer at 650 nm and 280 nm, respectively. The ratio of Cy5 to diabody was calculated to confirm the number of fluorophore molecules conjugated to each diabody molecule.

Sonn G.A., Behesnilian A.S., Jiang Z.K., Zettlitz K.A., Lepin E.J., Bentolila L.A., Knowles S.M., Lawrence D., Wu A.M, & Reiter R.E. (2015). Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real-Time. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(6), 1403-1412.

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Biotinylation of Antibodies and IgG

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BioAbs, except for Bio-CD133, for AvIR-mediated PIT were chemically prepared using EZ-Link Sulfo-LC-NHS-Biotinylation Kit (Thermo Fisher Scientific) following manufacturer’s instructions. A Zeba Desalt Spin Column (Thermo Fisher Scientific) was used to remove excess biotin reagent and exchange the buffer with Dulbecco’s phosphate-buffered saline (DPBS). We also performed a biotinylation of human immunoglobulin G (IgG) for using as an irrelevant control (Bio-hIgG).

Shirasu N., Shibaguchi H., Yamada H., Kuroki M, & Yasunaga S. (2019). Highly versatile cancer photoimmunotherapy using photosensitizer-conjugated avidin and biotin-conjugated targeting antibodies. Cancer Cell International, 19, 299.

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Fluorescent Labeling of Protein Nanoparticles

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For fluorescent visualization, SI (control ELP) and crySI were covalently modified with NHS-Fluorescein (Thermo Fisher Scientific Inc., Rockford, IL) by conjugation to free amines. Briefly, crySI was mixed with a 3-fold molar excess of NHS-Fluorescein in phosphate buffered saline (PBS) and incubated at 4 °C for 3 h. Free fluorophore was removed by size exclusion chromatography using a Zeba desalt spin column (89891, Thermo Fisher Scientific Inc., Rockford, IL). The concentration of label after the purification, C_FL-crySI, was estimated as follows:

C_{F L - c r y S I} = \frac{A_{493}_{n m}}{ε_{f l u o r}}

where the molar extinction coefficient of fluorescein, E_fluor, was assumed to be 70,000 M⁻¹ cm⁻¹. Due to the low molar extinction coefficient of the crySI relative to the contribution of fluorescein at an optical absorbance of 280 nm, the degree of labeling, N_labeling, was estimated as follows:

N_{l a b e l i n g} = \frac{n_{F L - c r y S I, p u r i f i e d}}{n_{c r y S I, r e a c t e d}}

where n_{crySI,reacted} and n_{FL-crySI,purifled} are the moles of crySI reacted and fluorescein recovered after purification respectively. Fluorescein labeled FL-crySI had a labeling efficiency ~0.6. To determine the purity of labeled materials, proteins were separated on SDS-PAGE gels and imaged on a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA).

Sreekumar P.G., Li Z., Wang W., Spee C., Hinton D.R., Kannan R, & MacKay J.A. (2018). Intra-vitreal αB crystallin fused to elastin-like polypeptide provides neuroprotection in a mouse model of age-related macular degeneration. Journal of controlled release : official journal of the Controlled Release Society, 283, 94-104.

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Preparation of DDM-Solubilized Membrane Proteins

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n-Dodecyl-β-d-maltoside (DDM) was purchased from Dojindo Laboratories (Kumamoto,
Japan). UQ-10, dithiothreitol (DTT), and trans-4,5-dihydroxy-1,2-dithiane
(oxidized DTT) were purchased from Wako Pure Chemical Industries Ltd.
(Osaka, Japan). UQ-1 was purchased from Sigma-Aldrich (St. Louis,
MO). NeutrAvidin, Zeba Desalt Spin Column, and tris(2-carboxyethyl)phosphine
(TCEP) were obtained from Thermo Fisher Scientific Inc. (Yokohama,
Japan). Maleimide–poly(ethylene glycol) (malPEG, SUNBRIGHT
ME-050MA, 5 kDa) was purchased from NOF Corporation (Tokyo, Japan).
Unless stated otherwise, all other reagents were purchased from Nacalai
Tesque Co. (Kyoto, Japan). Type 1 ultrapure water (MilliQ water, Merck
Ltd., Tokyo, Japan) was used in all experiments.

Yazawa K, & Furusawa H. (2019). Entropy-Driven Mechanisms between Disulfide-Bond Formation Protein A (DsbA) and B (DsbB) in Escherichia coli. ACS Omega, 4(5), 8341-8349.

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Quantitative Analysis of RhoA Dynamics

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Human breast cancer cell line MDA-MB-231 was purchased from ATCC. RhoA antibody (sc-418) and donkey anti-mouse IgG-FITC (sc-2099) were purchased from Santa Cruz. Duramycin, biotin and phalloidin tetramethylrhodamine (Cat#P1951) were from Sigma; biotinylated Duramycin was synthesised as described earlier (8 (link)); avidin-Texas red conjugate (A820) and avidin-FITC (Cat#43-4411) were from Life Technologies; Zeba desalt spin column (Cat#89890) was from Thermo Scientific. GFP-tagged wild-type RhoA (GFP-RhoA), constitutively active RhoA (GFP-CA-RhoA), wild-type RhoA with positive charges in the PBR mutated to Q (GFP-RhoA-PBRQ) and constitutively active RhoA with positive charges in the PBR mutated to Q (GFP-CA-RhoA-PBRQ) were prepared and used as previously described (9 (link)). Lact-C2-GFP (Cat#22852), 2PH-PLCdelta-GFP (Cat#35142) and PM-GFP (Cat#21213) were purchased from Addgene. LifeAct–TagGFP2 (Cat#60101) was purchased from Ibidi.

Hou S., Grillo D., Williams C.L., Wasserstrom J.A., Szleifer I, & Zhao M. (2014). Membrane phospholipid redistribution in cancer micro-particles and implications in the recruitment of cationic protein factors. Journal of Extracellular Vesicles, 3, 10.3402/jev.v3.22653.

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Quantitative Anti-drug Antibody Assay

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The MEDI7247 reference standard material (Ref) and the purified main peak (MP) and pre-peak (PP) materials, and the capture reagents, including two anti-idiotope antibodies (anti-IDs) and one anti-PBD antibody (anti-PBD), were provided by AstraZeneca (Gaithersburg, MD, USA). The peptides were synthesized at New England Peptide (Gardner, MA, USA). SMART IA beads, EZ-Link^® Sulfo-NHS-LC-Biotin, a Zeba™ Desalt Spin Column, acetonitrile (ACN) and formic acid (FA) were obtained from Thermo Scientific (Waltham, MA, USA). The enzymes, including IdeS, trypsin and chymotrypsin, were from Promega (Madison, WI, USA). The Rapigest, Acquity BEH C18 columns and HLB solid-phase extraction (SPE) plates were purchased from Waters (Middleton, MA, USA). LCMS-grade water was purchased from JT Baker. Protein LoBind vials and plates (Eppendorf, Hamburg, Germany) were used for all LCMS sample preparation. General reagents, buffers and plates were purchased from VWR (Radnor, PA, USA). Selected samples from patients participating in clinical study NCT03106428 were analyzed using the method described. This study was conducted in accordance with principles of the Declaration of Helsinki and the International Conference on Harmonization Guidance for Good Clinical Practice. Independent ethics committee approval was obtained.

Huang Y., Yuan J., Mu R., Kubiak R.J., Ball K., Cao M., Hussmann G.P., de Mel N., Liu D., Roskos L.K., Liang M, & Rosenbaum A.I. (2023). Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate. Antibodies, 12(4), 66.

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