Biotin-maleimide, DBCO-PEG4-biotin and N-succinimidyl-S-acetyl-thioacetate (SATA) were from Sigma-Aldrich (Saint Louis, Missouri, USA). Luminol, Nunc 96-well maxisorp microtiter plates and Zeba Spin 7 kDa desalting columns were from Thermo Scientific (Waltham, Massachusetts, USA). Spectramax L and Spectramax M2 were from Molecular Devices (San Jose, California, USA). Streptavidin-polyHRP and secondary rabbit anti-mouse HRP antibody were from Agilent Technologies (Santa Clara, California, USA). BL21 pLyss E. Coli was obtained from Invitrogen (Carlsbad, California, USA) and vector pet30b-7M SrtA (plasmid # 51141), encoding a modified, calcium-independent Sortase A from S. aureus was a gift from Hidde Ploegh and ordered from Addgene (Watertown, Massachusetts, USA). DBCO-PEG (20 kDa) was from Jena Bioscience (Jena, Germany) and mPEG-maleimide (20 kDa) was from Broadpharm (Waples, San Diego, USA). DABCYL-LPETG-EDANS was from AnaSpec (Waddinxveen, the Netherlands). Gly3-azide was from IRIS Biotech GmbH (Marktredwitz, Germany). Imidazole was from Merck Millipore (Burlington, Massachusetts, USA). Phe-Pro-Arg-chloromethylketone (PPACK) was from Haematologic Technologies (Essex Junction, Vermont, USA). Talon Superflow was from GE Healthcare (Hoevelaken, The Netherlands).
N succinimidyl s acetylthioacetate sata
Manufactured by Merck Group
Sourced in United States
N-succinimidyl S-acetylthioacetate (SATA) is a heterobifunctional cross-linker used in biochemistry and molecular biology. It contains an N-hydroxysuccinimide (NHS) ester group and a protected thiol group, which allows for the covalent attachment of molecules to amine-containing compounds.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m2, by Molecular Devices (1 mentions)
Luminol, by Thermo Fisher Scientific (1 mentions)
Imidazole, by Merck Group (1 mentions)
Biotin maleimide, by Merck Group (1 mentions)
Dbco peg4 biotin, by Merck Group (1 mentions)
Dabcyl lpetg edans, by AnaSpec (1 mentions)
Talon superflow, by GE Healthcare (1 mentions)
Spectramax l, by Molecular Devices (1 mentions)
Phe pro arg chloromethylketone ppack, by Prolytix (1 mentions)
Gly3 azide, by Iris Biotech (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cholesterol chol, by Merck Group (1 mentions)
Quant it ribogreen rna assay, by Thermo Fisher Scientific (1 mentions)
G 25 sephadex quick spin protein columns, by Roche (1 mentions)
4 protocols using n succinimidyl s acetylthioacetate sata
1
Sortase A-Mediated Protein Labeling
2
Preparation of Lipid Nanoparticles for siRNA Delivery
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Lipids dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3) were obtained from Med Chem Express (Monmouth Junction, NJ, USA). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (poly-ethylene glycol)-2000]-maleimide (DSPE-PEG-Mal) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). 1,1′-Diooctadecyl-3,3,3′,3′-teramethyl–indocarbocyanine perchlorate (DiI) as a lipophilic tracker and Hoechst 33,342 as a nucleic acid stain were obtained from Molecular Probes (Leiden, The Netherlands). Cholesterol (Chol) and N-succinimidyl S-acetylthioacetate (SATA) were purchased from Sigma (St. Louis, MO, USA). EGFP-S1 positive control DsiRNA (siRNAGFP) was purchased from Integrated DNA Technologies Inc. (Coralville, IA, USA). Other siRNA were obtained from Qiagen (Venlo, The Netherlands). LipofectamineTM 2000 was obtained from Invitrogen (Breda, The Netherlands). The hybridoma for producing E1/6-aa2 monoclonal antibody (mouse IgG1 against human VCAM-1) was kindly given by Dr. M. Gimbrone from the Harvard Medical School (Boston, MA, USA).
3
Chemical Labelling of Biomolecules
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N-succinimidyl-S-acetylthioacetate (SATA) was obtained from Sigma (St. Louis, MO, USA). Nucleic acid stain Hoechst 33342 (trihydrochloride) and lipophilic tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) were obtained from Molecular Probes (Leiden, The Netherlands).
4
LNP Conjugation with mAbs for PECAM-1 and PLVAP
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LNP was conjugated with mAbs specific for platelet endothelial cell adhesion molecule-1 (PECAM-1) or plasmalemma vesicle-associated protein (PLVAP). Anti-mouse PLVAP antibody was produced in-house from hybridoma secreting rat anti-mouse PLVAP IgG2a, clone MECA32, which was obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained at the University of Iowa, Department of Biology). Anti-mouse-PECAM-1/CD31 antibody (clone MEC13, BioLegend, San Diego, CA), anti-mouse PLVAP antibody, or control isotype-matched IgG were coupled to LNP via SATA–maleimide conjugation chemistry, as described previously [9 (link)]. Briefly, LNP was modified with DSPE-PEG-maleimide by a post-insertion technique. The antibody was modified with N-succinimidyl S-acetylthioacetate (SATA) (Sigma-Aldrich) to introduce sulfhydryl groups allowing conjugation to maleimide. SATA was deprotected using 0.05 M hydroxylamine followed by removal of the unreacted components by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN). The reactive sulfhydryl group on the antibody was then conjugated to maleimide moieties using thioether conjugation chemistry. Purification was performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich). mRNA content was calculated by performing a modified Quant-iT RiboGreen RNA assay (Invitrogen).
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