Sulfo sda

The cross-linker sulfo-SDA (sulfosuccinimidyl 4,4′-azipentanoate) (Thermo Scientific) was dissolved in cross-linking buffer (10 mM HEPES pH 7.8, 150 mM NaCl, 4 mM MgCl₂, 0.5 mM TCEP) to 100 mM before use. The labelling step was performed by incubating 18 μg aliquots of the complex at 1 mg/mL with 2, 1, 0.5, 0.25, 0.125 mM sulfo-SDA, added, respectively, for an hour. The samples were then irradiated with UV light at 365 nm, to form cross-links, for 20 min and quenched with 50 mM NH₄HCO₃ for 20 min. All steps were performed on ice. Reaction products were separated on a Novex Bis-Tris 4–12% SDS−PAGE gel (Life Technologies). The gel band corresponding to the cross-linked complex was excised and digested with trypsin (Thermo Scientific Pierce) [116 (link)] and the resulting tryptic peptides were extracted and desalted using C18 StageTips [117 (link)]. Eluted peptides were fractionated on a Superdex Peptide 3.2/300 increase column (GE Healthcare) at a flow rate of 10 μL/min using 30% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid as mobile phase. 50 μL fractions were collected and vacuum-dried.

85 μg of ScElongator complex at 2.22 μM concentration was crosslinked with sulfo-SDA (sulfosuccinimidyl 4,4′-azipentanoate, Thermo Scientific) at 1:500 and 1:1000 sample:crosslinker molar ratios. The reaction was incubated at room temperature for 60 min irradiated with UV light at 365 nm using a Luxigen34 LZ1 LED emitter (Osram Sylvania Inc.) for 10 s, and quenched with a final concentration of 50 mM ammonium bicarbonate (ABC). The crosslinked complex was separated from single subunits on a Novex Bis–Tris 4–12% SDS-PAGE gel (Life Technologies). Gel pieces containing the crosslinked complex were excised. The sample was reduced with DTT and free sulfhydryl groups were alkylated using iodoacetamide (74 (link)). Proteins were digested overnight at 37°C with 1 μg trypsin (Thermo Scientific Pierce) per 20 μg of protein sample. The digested peptides were recovered and desalted using C18 StageTips (75 (link)). The eluates of the StageTips were pooled and fractionated by size exclusion chromatography (SEC) using a Superdex™ 30 Increase 3.2/300 column (GE Healthcare) and a mobile phase consisting of 30% v/v ACN and 0.1% v/v trifluoric acid at a flow rate of 10 μl/min. The first six fractions (50 μl each) containing peptides were collected and the solvent was removed using a vacuum concentrator.

85 µg of HsElongator at 1.4 µM concentration were crosslinked with sulfo-SDA (sulfosuccinimidyl 4,4′-azipentanoate, Thermo Scientific) at 1:500 and 1:1000 protein:crosslinker molar ratios. For MmElongator, 60 µg at 5.0 µM concentration were used. After incubation at room temperature for 60 min, the reaction mix was irradiated with UV light at 365 nm using a Luxigen34 LZ1 LED emitter (Osram Sylvania Inc.) for 10 s. 50 mM ammonium bicarbonate was added to quench the reaction. SDS-PAGE was used to separate the crosslinked complexes from single subunits (Novex Bis-Tris 4–12% SDS−PAGE gel, Life Technologies), and gel sections containing the crosslinked complex were excised. Dithiothreitol reduced the sample and iodoacetamide alkylated free sulfhydryl groups^{70 (link)}. Proteins were digested using 1 µg trypsin (Thermo Scientific Pierce) per 20 µg of protein sample. The resulting peptides from the digestion were subjected to desalting using C18 StageTips^{71 (link)}.
The eluates from the StageTips were fractionated via size exclusion chromatography (SEC) using a Superdex™ 30 Increase 3.2/300 column (GE Healthcare). A mobile phase comprising 30% (v/v) acetonitrile (ACN) and 0.1% trifluoroacetic acid at a flow rate of 10 µl/min was used. The first six 50 µL fractions containing peptides were collected and the solvent was removed using a vacuum concentrator.