Methanol, acetic acid, and acetonitrile were purchased from Mallinckrodt (Phillipsburg, NJ). The peptide AADAADAA was custom synthesized by NeoBioLab (Cambridge, MA). The (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and ethylenediaminetetraacetic acid tetrasodium salt dihydrate were obtained from Sigma Aldrich (St. Louis, MO). The bis(sulfosuccinimidyl) suberate (BS3) cross-linker was obtained from Thermo Fisher Scientific Inc.(Rockford, IL). The N-hydroxysuccinimide ester of 4-trimethylammonium butyrate (TMAB-NHS) reagent was a generous donation from Prof. Fred Regnier. All peptide solutions for positive nanoelectrospray (nESI) were prepared in 49.5/49.5/1 (v/v/v) Methanol/water/acetic acid, while the peptide solutions for negative nESI were mixed in 49.5/49.5/1 (v/v/v) Methanol/water/ammonium hydroxide solution (~50 μM). The BS3, ethylenediaminetetraacetic acid (EDTA) and (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6-TCBA) reagents were dissolved in water (~2 mM), while the TMAB-NHS was dissolved in acetonitrile (~2 mM).
Bis sulfosuccinimidyl suberate bs3 cross linker
Manufactured by Thermo Fisher Scientific
Bis(sulfosuccinimidyl) suberate (BS3) is a water-soluble, homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester cross-linking agent. It is used to covalently link primary amine groups, such as those found on the side chains of lysine residues in proteins.
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2 protocols using bis sulfosuccinimidyl suberate bs3 cross linker
1
Peptide Crosslinking and Characterization
2
Cbp1-Cren7 Crosslinking Assay
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Cbp1 and Cren7 aliquots were buffer-exchanged into 25 mM HEPES/NaOH pH 7.5, 200 mM NaCl by ultrafiltration. DNA Templates were annealed in 10 mM HEPES/NaOH pH 7.5, 50 mM NaCl. Cbp1, Cren7, and template DNA concentrations were kept at 2.5 μM. Samples were incubated at 75 °C for 5 min. 1 mM bissulfosuccinimidyl suberate (BS3) cross-linker (ThermoFisher) was added to each sample and incubated at 25 °C for 30 min. Cross-linking reactions were quenched by the addition of 50 mM Tris/HCl (pH 8.0) and samples were resolved by SDS-PAGE followed by Coomassie Blue staining.
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