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Nap 10 sephadex g 25 column

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The NAP-10 Sephadex G-25 column is a disposable, pre-packed column used for desalting and buffer exchange of small molecules, proteins, and peptides. The column is filled with Sephadex G-25 resin, which provides efficient separation and removal of salts, buffers, and other low molecular weight compounds from sample solutions.

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Lab products found in correlation

Glomax multi plate reader, by Promega (2 mentions) Glomax multi, by Promega (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions)

Close spelling variants of this product

NAP-25 column Sephadex® G-25 NAP-10 column Sephadex G-10 G-25 column NAP-25 NAP-10 NAP column Sephadex columns

6 protocols using nap 10 sephadex g 25 column

1

Calcein Leakage Assay for Lipid Vesicles

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Purified calcein-loaded LUVs were mixed with D-dfTAT in presence or absence of UNC7938 (1–100 μM) at a 1:50 peptide: lipid ratio for 1 h at room temperature in 100 mM NaCl, 10 mM NaH2PO4 pH5.5. UNC7938 stock solution Samples were centrifuged for 1 min at 4,000 rpm. To measure the amount of leaked calcein and separate soluble liposomes from released calcein, the supernatants were purified using an illustra NAP-10 Sephadex G-25 column (GE Healthcare) (the elution volumes of liposomes and free calcein were determined independently with pure samples). Fractions were collected in a 96-well plate and the fluorescence of calcein was measured using a Promega GloMax-Multi plate reader (Ex 490nm, Em 520–560nm). Note, since UNC7938 stock is dissolved in 100% DMSO. The control of D-dfTAT+ 0.1% DMSO (concentration of DMSO in the UNC7938 incubations) with liposomes was performed as a control. 100% leakage was established by treating liposomes with the detergent Triton X-100.
Allen J., Najjar K., Erazo-Oliveras A., Kondow-McConaghy H.M., Brock D.J., Graham K., Hager E.C., Marschall A.L., Dübel S., Juliano R.L, & Pellois J.P. (2019). Cytosolic delivery of macromolecules in live human cells using the combined endosomal escape activities of a small molecule and cell penetrating peptides. ACS chemical biology, 14(12), 2641-2651.
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2

Conjugation of scFvs to Tetrazine-Peptide

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Example 25

This example aimed to demonstrate that three scFvs could be conjugated to the three PEG12-maleimide linking arms based on tetrazine-peptide 2. Prior to conjugation with the tetrazine-peptide 2 that had three PEG12-maleimide linking arms, 1F10 scFv was incubated with DTT at a molar ratio of 2:1 ([DTT]:[scFv]) at 25° C. for 4 hours with gentle shaking to keep its C-terminal cysteine in reduced form. Subsequently, the buffer of reduced 1F10 scFv was exchanged to maleimide-SH coupling reaction buffer (100 mM sodium phosphate, pH 7.0, 50 mM NaCl and 5 mM EDTA) by using an NAP-10 Sephadex G-25 column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the tetrazine-peptide 2 having three PEG12-maleimide linking arms was conducted overnight at 4° C. at a molar ratio of 1:4 ([linker]:[Protein]).

US09907858B2. Peptide core-based multi-arm linkers (2018-03-06). Immunwork Inc. [TW]. Inventors: Tse-Wen Chang [TW], Hsing-Mao Chu [TW].
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3

Conjugation of scFv to Tetrazine-Peptide

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Example 26

This example aimed to demonstrate that three scFvs could be conjugated to the three PEG12-maleimide linking arms based on tetrazine-peptide 2. Prior to conjugation with the tetrazine-peptide 2 that had three PEG12-maleimide linking arms, 1F10 scFv was incubated with DTT at a molar ratio of 2:1 ([DTT]:[scFv]) at 25° C. for 4 hours with gentle shaking to keep its C-terminal cysteine in reduced form. Subsequently, the buffer of reduced 1F10 scFv was exchanged to maleimide-SH coupling reaction buffer (100 mM sodium phosphate, pH 7.0, 50 mM NaCl and 5 mM EDTA) by using an NAP-10 Sephadex G-25 column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the tetrazine-peptide 2 having three PEG12-maleimide linking arms was conducted overnight at 4° C. at a molar ratio of 1:4 ([linker]:[Protein]).

US10010626B2. Molecular constructs with targeting and effector moieties (2018-07-03). Immunwork Inc. [TW]. Inventors: Tse-Wen Chang [TW], Hsing-Mao Chu [TW], Chun-Yu Lin [TW].
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4

Calcein Leakage Assay for Peptide-Lipid Interactions

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Purified calcein-loaded LUVs were mixed with different peptides at a 1:50 peptide: lipid ratio for 1 h at room temperature in 100 mM NaCl, 10 mM NaH2PO4 pH5.5. Samples were centrifuged for 1 min at 4,000 rpm. To measure the amount of leaked calcein and to separate soluble liposomes from released calcein, the supernatants were purified using an illustra NAP-10 Sephadex G-25 column (GE Healthcare) (the elution volumes of liposomes and free calcein were determined independently with pure samples). Fractions were collected in a 96-well plate and the fluorescence of calcein was measured using a Promega GloMax-Multi plate reader (Ex 490nm, Em 520–560nm). The percent leakage was calculated using the following equation:
%Leakage=100×Flt-Fl0Flmax-Fl0 where Flt is the free calcein fluorescence intensity of a sample at a specific peptide:lipid ratio measured after 1 h, Fl0 is the free calcein fluorescence intensity of an untreated sample and Flmax is the free calcein fluorescence intensity of a sample after treatment with 0.2 % Triton X-100.
Najjar K., Erazo-Oliveras A., Mosior J.W., Whitlock M.J., Rostane I., Cinclair J.M, & Pellois J.P. (2017). Unlocking endosomal entrapment with supercharged arginine-rich peptides. Bioconjugate chemistry, 28(12), 2932-2941.
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5

Calcein Leakage Assay for Liposome Permeabilization

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A calcein-leakage assay was employed to evaluate the permeabilization of liposomes by dfTAT. For this assay, LUVs of different lipid compositions containing 60 mM calcein in their lumen were prepared. LUVs were mixed with dfTAT and fTAT at different peptide:lipid ratios in phosphate buffer (100 mM NaCl, 10 mM NaH2PO4 pH 5.5). After 60 min, the amount of free calcein was established by purifying the supernatants obtained using an illustra NAP-10 Sephadex G-25 column (GE Healthcare). The supernatants obtained after low speed centrifugation were purified to exclude soluble liposomes. The fluorescence from free calcein, from the fractions collected, was measured using the blue channel (Ex 490 nm, Em 520–560 nm) of a Promega GloMax-Multi plate reader (Promega). The extent of liposomal lumen content release or % leakage was determined according to the following equation:
%Leakage=100×Flt−Fl0Flmax−Fl0 Where Flt is the free calcein fluorescence intensity of a sample at a specific peptide:lipid ratio measured after 60 min, Fl0 is the free calcein fluorescence intensity of a untreated sample and Flmax is the free calcein fluorescence intensity of a sample after treatment with 0.2 % Triton X-100.
Erazo-Oliveras A., Najjar K., Truong D., Wang T.Y., Brock D.J., Prater A.R, & Pellois J.P. (2016). The late endosome and its lipid BMP act as gateways for the efficient cytosolic access of the delivery agent dfTAT and its macromolecular cargos. Cell chemical biology, 23(5), 598-607.
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6

Functionalization of MWCNT with CS-ODN

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25 µl of a 1 mM NH2-CS-ODN solution in water was mixed with 7.5 µl NaHCO3-buffer (pH 8.6, 1 M) and 25 µl DSS (10 mg/ml in DMSO). Excess DSS was separated using an illustra™ NAP™-10 sephadex™ G-25 column (GE Healthcare, Munich, Germany) and activated ODN were concentrated by Amicon® Ultra centrifugal filters (3000 MWCO; Merck, Darmstadt, Germany). 0.5 nmol of the activated NH2-CS-ODN were mixed with 50 µg MWCNT-NH2 in 0.5 ml PBS and shaken at room temperature overnight followed by the removal of the excess CS-ODN.
Kaufmann A., Hampel S., Rieger C., Kunhardt D., Schendel D., Füssel S., Schwenzer B, & Erdmann K. (2017). Systematic evaluation of oligodeoxynucleotide binding and hybridization to modified multi-walled carbon nanotubes. Journal of Nanobiotechnology, 15, 53.
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