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Nap 5 sephadex g 25 column

Manufactured by GE Healthcare
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The NAP-5 Sephadex G-25 columns are size exclusion chromatography columns designed for desalting and buffer exchange of small molecules. The columns are packed with Sephadex G-25, a cross-linked dextran gel, which allows for the separation of molecules based on their size and molecular weight.

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Lab products found in correlation

Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions) His select spin columns, by Merck Group (1 mentions) Cf568, by Biotium (1 mentions) Anti actin antibody, by Thermo Fisher Scientific (1 mentions) 15 s hete, by Cayman Chemical (1 mentions) 15 s hpete, by Cayman Chemical (1 mentions)

Close spelling variants of this product

NAP-5 Sephadex G-25 DNA Grade columns NAP-25 column Sephadex® G-25 NAP-5 column G-25 column NAP-25 NAP column NAP-5 Sephadex columns

5 protocols using nap 5 sephadex g 25 column

1

DBCO Reagent Conjugation to Antibodies

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DBCO reagents, DBCO-CR110, DBCO-Cy5, and DBCO-Biotin were reconstituted in DMSO at 1 mg/ml, whereas DBCO-mPEG 5 kDa was reconstituted at 5 mg/ml (Click Chemistry Tools catalog nos. A118-25, A127-25, A130-1, and A116-10). For SPAAC conjugations, antibodies at a final concentration of 4 mg/ml were incubated with 6-fold molar excess of DBCO reagents in PBS, pH 7.2, containing 10% DMSO at room temperature for 20–24 h (22 (link)). Unreacted DBCO reagents were removed using an illustra NAP-5 Sephadex G-25 column (GE Healthcare catalog no. 17-0853-02) pre-equilibrated with PBS. Conjugation efficiency was evaluated using rLC/MS for all antidotes, and DBCO-mPEG conjugation was also evaluated by SDS-PAGE (Fig. 4).
Portnoff A.D., Gao C., Borrok M.J., Gao X., Gao C, & Rainey G.J. (2017). An antidote approach to reduce risk and broaden utility of antibody-based therapeutics. The Journal of Biological Chemistry, 292(20), 8498-8506.
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2

N2OR Turnover Assay Protocol

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Turnover assays were conducted using activated N2OR with 40 and 65 % of CuZ(4Cu2S). Activated enzyme (after 3 h incubation with reduced methyl viologen in 100 mM Tris-HCl pH 7.6, inside an anaerobic chamber) was desalted using a NAP-5 Sephadex G25 column (GE Healthcare) equilibrated with 100 mM potassium phosphate pH 7.0 [29] . The collected enzyme was concentrated using an ultrafiltration micro concentrator (MW cut off 30 kDa) to a final concentration of 124 μM. A drop of this activated N2OR (4 μL) was immobilized on the MWCNT-GCE, using the procedure described before. Turnover assays were also performed in 100 mM potassium phosphate pH 7.0 electrolyte solution, in the presence of increasing concentrations of N2O-saturated water, up to 1.4 mM.
Carreira C., Dos Santos M.M.C., Pauleta S.R, & Moura I. (2020). Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide reductase from Marinobacter hydrocarbonoclasticus - An electrochemical study. Bioelectrochemistry (Amsterdam, Netherlands), 133.
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3

Cloning, Expression, and Purification of Cbl-b and Tyro3 Kinases

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Human Cbl-b TKB+RF (residues 38–429) and Tyro3 kinase catalytic domain (residues 495–810) were cloned into pET-24d(+)-based vectors for bacterial expression. Tyro3 isolated from E. coli was found to be in a phosphorylated state, possibly due to autophosphorylation. Cbl-b point mutations (Y106F, Y133F, and Y363F), in addition to a compound mutant (Y106/133/363F) were generated from plasmid pE-10xHis-HA-Cbl-b TKB+RF by site-directed mutagenesis. WT and mutant Cbl-b TKB+RF proteins were purified from IPTG-induced bacteria transformed with the various constructs. The cells were lysed and sonicated for 30 seconds x 3 cycles, loaded onto HIS-select spin columns (Sigma), and purified according to the manufacturer’s protocol. To remove the imidazole contained in the elution buffer prior to assays, buffer exchange was performed using NAP-5 Sephadex G-25 columns (GE Healthcare) using 20 mM Tris HCl pH 8.0. Proteins were stored in 150 mM NaCl, 10% glycerol, and 10mM DTT for stability. Protein concentration was measured using Pierce 600 nm kit (Thermo Scientific).
Chirino L.M., Kumar S., Okumura M., Sterner D.E., Mattern M., Butt T.R, & Kambayashi T. (2019). TAM receptors attenuate murine NK cell responses via E3 ubiquitin ligase Cbl-b. European journal of immunology, 50(1), 48-55.
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4

Fluorescent Antibody Labeling Protocol

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CF 680 and CF 658 secondary antibodies were generated by incubating 100-μg IgG Fab fragments dissolved in in NaHCO3 (50 mM, pH 8.1) with a 5-fold excess of succinimidyl esters of fluorescent dye CF 680 or CF 568 dyes (Biotium, USA) in DMSO (10 μM) for 1 hour at RT under gentle agitation. Unbound dye was removed with Nap-5 Sephadex G-25 columns (GE Healthcare) and labelled antibody eluted from the column with PBS.
Schmerl B., Gimber N., Kuropka B., Stumpf A., Rentsch J., Kunde S.A., von Sivers J., Ewers H., Schmitz D., Freund C., Schmoranzer J., Rademacher N, & Shoichet S.A. (2022). The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses. PLoS Biology, 20(3), e3001503.
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5

Purification and Analysis of PTEN and its Interactions

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Recombinant wild-type PTEN was purified as described previously [30 (link)]. 15s-HpETE and 15s-HETE were purchased from Cayman Chemical (Ann Arbor, MI, USA). NAP-5 Sephadex G25 columns were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Lipofectamine 2000 transfection reagent, and anti-actin antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The PTEN and Trx antibodies were prepared as described previously [39 (link), 40 (link)]. Anti-rabbit IgG horseradish peroxidase-conjugated antibody was purchased from Ab Frontier (Daejeon, Korea).
Zhang Y., Park J., Han S.J., Lim Y., Park I., Kim J.S., Woo H.A, & Lee S.R. (2019). Peroxiredoxin III Protects Tumor Suppressor PTEN from Oxidation by 15-Hydroperoxy-eicosatetraenoic Acid. Oxidative Medicine and Cellular Longevity, 2019, 2828493.
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