Colloidal blue

Eluted immunecomplexes were run in two parallel lanes of a precast NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and visualized with Colloidal Blue (Invitrogen). Both gel lanes were separately cut into slices and subjected to in-gel trypsinization (Promega) as previously described53 (link). Briefly, gel slices were subjected to reduction with 10 mM DTT, alkylation by 55 mM chloroacetamide and protein digestion by incubating with trypsin overnight at 37 °C.
Resulting tryptic peptides were extracted from the gel by serial incubations with 100% ACN and 30% ACN/3% TFA. Finally, the solutions obtained in all the incubations were pooled together and dried down in a vacuum centrifuge. Whereas peptides derived from one of the lanes were directly concentrated and desalted using C₁₈ stage tips (made in house using Empore disc –C18 Agilent Life Science) to further analyze by LC-MS/MS, peptides derived from the other lane were subjected to TiO₂ enrichment prior to MS analysis.

The immunoprecipitates were run on an SDS-PAGE gel (NuPAGE® Novex 4–12% Bis-Tris gel; Invitrogen, Carlsbad, CA, USA) followed by staining with Colloidal Blue (Invitrogen). The SDS-PAGE gel was sliced into eight pieces for in-gel tryptic digestion using an in-gel tryptic digestion kit (Thermo Fisher Scientific, Rockford, IL, USA), according to the manufacturer's instructions. Briefly, the excised gels were destained, reduced using Tris [2-carboxyethyl] phosphine (TCEP) and alkylated using idoacetamide (IAA). The alkylated gel pieces were dehydrated in 100% acetonitrile (ACN) and digested with mass spectrometry (MS) grade trypsin in 25 mM NH₄CO₃ for 12 h at 30°C. The digested peptides were evaporated using a vacuum concentrator and cleaned using C18 spin columns (Thermo Fisher Scientific) for MS analysis.

Each cell pellet was lysed in B-per supplemented with lysozyme, Dnase I, and EDTA using the B-PER Kit (Pierce, Thermo Fisher Scientific) following manufacturers’ instructions. Protein concentrations of the cell lysates were determined with the BCA protein assay (Pierce, Thermo Fisher Scientific). Twenty micrograms (20 µg) of protein from each sample was diluted in LDS PAGE buffer (Invitrogen) followed by reducing, heat denaturing, and separation on a 10% SDS Bis-Tris gel (Invitrogen). The gel was stained overnight with Colloidal Blue (Invitrogen), and the two most abundant bands, A and B (Figure 1), were first carefully excised. Based on staining intensities, the rest of the gel lane was then cut into six nearly equal fractions from the top to bottom, and all eight of the resultant gel bands were then equilibrated in 100 mM ammonium bicarbonate (AmBc). Gel slices were reduced, carbidomethylated, dehydrated, and digested with Trypsin Gold (Promega) as per manufacturers’ instructions. Following digestion, peptides were extracted, volumes were reduced in a SpeedVac to near dryness, and re-suspended to 20 µL using 95% ddH₂O/5% ACN/0.1% formic acid (FA) prior to analysis by 1D reverse phase LC-ESI-MS2 (as outlined below).