Nhs activated sepharose fast flow

Manufactured by GE Healthcare

NHS-activated Sepharose Fast Flow is a pre-activated agarose-based medium designed for covalent immobilization of ligands containing primary amino groups. The NHS-ester functional groups on the medium's surface facilitate the coupling of ligands under mild conditions. This product is suitable for use in affinity chromatography, enzyme immobilization, and other biochemical applications.

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Lab products found in correlation

Benzonase, by Merck Group (2 mentions) Lysyl endopeptidase lys c, by Fujifilm (1 mentions) Dbco amine, by Merck Group (1 mentions) Dibenzocyclooctyne peg4 biotin conjugate, by Merck Group (1 mentions) Af488 azide, by Jena Biosciences (1 mentions) Trypsin gold, by Promega (1 mentions) Edta free protease inhibitor, by Roche (1 mentions)

3 protocols using nhs activated sepharose fast flow

Antibody-based Protein Purification and Detection

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The anti-Gea2 rabbit polyclonal antibody was generated using purified Gea2 GEF domain (Covance) and used at a 1:500 dilution. This antibody cross-reacts to some extent with purified recombinant Gea1 but fails to detect Gea1 in yeast extracts. The anti-G6PDH rabbit polyclonal antibody was purchased from Sigma and used at a 1:30,000 dilution. The anti-His₆ mouse monoclonal antibody (mAb) used to verify cleavage of His tags was purchased from Covance and used at a 1:500 dilution.
The GFP nanobody resin used for pull downs in Figures 3 and 4 was made using purified GFP nanobody and NHS-activated Sepharose Fast Flow (GE Healthcare) (Kirchhofer et al., 2010 (link)). Twenty-five ODs of cells were suspended in lysis buffer (50 mM Tris–HCl, pH 7.5, 0.2% NP40 substitute, 150 mM NaCl, 1 mM EDTA, 1× complete protease inhibitor cocktail [Roche] and 1 mM phenylmethylsulfonyl fluoride [PMSF]) before lysis by mechanical disruption. After incubation, resin was washed three times with lysis buffer, and proteins were eluted in SDS sample buffer.

Gustafson M.A, & Fromme J.C. (2017). Regulation of Arf activation occurs via distinct mechanisms at early and late Golgi compartments. Molecular Biology of the Cell, 28(25), 3660-3671.

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Purification and Labeling of Ribosome and Cas9

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Purified E. coli ribosome
(E. coli B strain) was purchased from New England
Biolabs (MA, USA) and diluted with dilution buffer (50 mM HEPES, 50
mM KCl, pH 7.5, 10 mM MgAc₂) to a concentration of 1 mg/mL.
Purified recombinant Cas9 from S. pyogenes fused
with a Halo-tag was generated in house, as described by Deng et al.^{25 (link)} DSBSO was synthesized similarly as described
by Burke et al.^{24 (link)} For enrichment similar
as described by Kaake et al.^{15 (link)} (BARAC Method),
Dibenzocyclooctyne-PEG4-biotin conjugate (#760749-5MG, Sigma-Aldrich)
was used without further purification. Biotin was pulled using Pierce
High Capacity Streptavidin Resin (# 20359, Thermo). DBCO beads were
synthesized in house: NHS-activated Sepharose fast flow (#17-0906-01,
GE Healthcare) was incubated to varying concentrations of dibenzocyclooctyne-amine
(DBCO-amine, #761540, Sigma-Aldrich). The prepared beads were stored
as 50% slurry in a 1:1 ethanol:water mixture. AF488-Azide (#CLK-1275-1,
Jena Bioscience) was used to test success of bead–DBCO coupling.
Trypsin gold was purchased from Promega (Mannheim, Germany) and lysyl
endopeptidase (LysC) was from Wako (Neuss, Germany). Benzonase—pharmaceutical
production purity—was from Merck (Darmstadt, Germany).

Matzinger M., Kandioller W., Doppler P., Heiss E.H, & Mechtler K. (2020). Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides. Journal of Proteome Research, 19(5), 2071-2079.

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GFP-tagged Protein Purification Protocol

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Transfected HEK293 cells were lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 2 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P40, EDTA-free protease inhibitors (Roche Applied Science), and 10 units of Benzonase (Sigma) with gentle rocking at 4°C for 1 h. Clarified lysate was incubated with anti-GFP conjugated resin prepared using NHS-activated Sepharose™ Fast Flow (GE Healthcare) for 1 h at 4°C. Resin was washed four times with 500 μl lysis buffer (without Benzonase). Resin-bound protein in 2× beta-mercaptoethanol and 2× SDS loading dye (Bio-Rad) was boiled and then loaded onto SDS-PAGE gels for identification by western blot.

Lo Y.H., Romes E.M., Pillon M.C., Sobhany M, & Stanley R.E. (2017). Structural Analysis Reveals Features of Ribosome Assembly Factor Nsa1/WDR74 Important for Localization and Interaction with Rix7/NVL2. Structure (London, England : 1993), 25(5), 762-772.e4.

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