Novex colloidal blue staining kit

For MS analysis, purified proteins were briefly separated by SDS-PAGE on Mini PROTEAN TGX gels (Bio-Rad), stained with the Novex colloidal blue staining kit (Invitrogen), and excised and further processed by in-gel Tryptic digestion. Peptides were desalted using C18 ZipTips (Millipore) and resuspended in 0.1% formic acid. LC-MS/MS was performed on a Q-Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher) with an Easy LC 1200 UPLC liquid chromatography system (Thermo Fisher) and analytical column ES803 (Thermo Fisher). Peptides were eluted with the following conditions at a flow rate of 300 nl min⁻¹: a 100-min gradient from 5% to 28% solvent B (80% [v/v] acetonitrile, 0.1% [v/v] formic acid), followed by a 20-min gradient from 28% to 44% solvent B and a short wash with 90% solvent B. Precursor scan was from mass-to-charge ratio (m/z) 375 to 1,600 and the top 20 most intense multiply charged precursors were selected for fragmentation with higher-energy collision dissociation (HCD) with normalized collision energy (NCE) 27.

Samples were prepared for analysis as follows: 20 μg of protein per sample in NuPAGE LDS sample buffer (Invitrogen) was loaded onto a Novex NuPage 10% Bis-Tris protein gel (Invitrogen), separated as a short stack, and stained overnight with Novex Colloidal Blue Staining kit (Invitrogen). Gels were then destained and lanes were cut into single molecular weight fractions. Following equilibration in 100 mM ammonium bicarbonate, fractions were digested overnight with Trypsin Gold (Mass Spectrometry grade (Promega)) and peptide extracts were reconstituted in 0.1% formic acid to 0.1 μg/μl.

Eight milliliters of cell culture media were concentrated and exchanged in PBS using Amicon Ultra 4 ml 3 kDa NMWL centrifugal unit (Millipore). Approximately 40 μg of protein per sample were then diluted to 35 μl using NuPAGE LDS sample buffer (Invitrogen). Proteins were reduced with DTT and denatured at 70 °C for 10 min prior to loading onto Novex NuPAGE 10% Bis-Tris Protein gels (Invitrogen). The gels were stained overnight with Novex Colloidal Blue Staining kit (Invitrogen). Following de-staining, each lane was cut into six MW fractions and equilibrated in 100 mM ammonium bicarbonate, each gel plug was then digested overnight with Trypsin Gold, Mass Spectrometry Grade (Promega) following manufacturer’s protocol. Peptide extracts were reconstituted in 0.1% Formic Acid/ddH2O at 0.1 μg/μl and used for LC-MS/MS analysis.

Triplicates of samples in each group were subjected to 1DE (50 μg/lane) and separated under reducing conditions on 10-well precast 4–12% Bis-Tris mini gels with 1x NuPAGE MES SDS Running Buffer (both from Thermo Fisher Scientific, Rockford, IL, USA). Gels were run for 60 min at 4 °C at a constant voltage of 150 V. Pre-stained protein standard, SeeBlue Plus 2 (Thermo Fisher Scientific, Rockford, IL, USA) was used as molecular mass marker and Novex Colloidal Blue Staining Kit (Invitrogen, Karlsruhe, Germany) was used to stain the gels according to the manufacturer's instructions. Subsequently, gels were destained overnight to eliminate background staining and scanned on Epson Perfection V600 Photo Scanner (Seiko Epson Corporation, Suwa, Nagano, Japan). Protein bands in each lane were excised (each gel lane were sliced into 20 pieces), reduced, alkylated and tryptic-digested, as described previously [19 (link),20 ]. In-gel tryptic-digested peptides were further purified using ZipTip C18 pipette tips (Millipore, Billerica, MA, USA), concentrated to dryness in a vacuum centrifuge and reconstituted with 0.1% trifluoroacetic acid (TFA) prior to LC-MS/MS analysis.