Novex nupage mops sds running buffer

IP’s were performed in quadruplicate. Following resuspension of the precipitates in Novex^™ NuPAGE^™ LDS sample buffer (Art. No. NP0007, Invitrogen^™), samples were incubated at 70°C for 10 min and separated on a Novex^™ NuPAGE^™ 4-12 % Bis-Tris Mini Protein Gel (Art. No. NP0321, Invitrogen^™) in 1x Novex^™ NuPAGE^™ MOPS SDS Running Buffer (Art. No. NP0001, Invitrogen^™) at 180 V for 10 min. After separation the samples were processed by in-gel digest as previously described ^{16 ,17} . Following protein digest, the peptides were desalted using a C18 StageTip ¹⁸ . For measurement the digested peptides were separated on a 25 cm reverse-phase capillary (75 μm inner diameter) packed with Reprosil C18 material (Dr. Maisch GmbH). Elution was carried out along a two hour gradient of 2 to 40 % of a mixture of 80 % acetonitrile/0.5 % formic acid with the EASY-nLC 1000 system (Art. No. LC120, Thermo Scientific^™). A Q Exactive^™ Plus mass spectrometer (Thermo Scientific^™) operated with a Top10 data-dependent MS/MS acquisition method per full scan was used for measurement ¹⁹ . Processing of the obtained results was performed with the MaxQuant software, version 1.5.2.8 against the Wormbase protein database (version WS263) for quantitation ²⁰ . The processed data was visualized with R^® and R-Studio^® using in-house scripts.