Tricine sample buffer

For RAW 264.7 cells, cells were washed with PBS and lysed in Tricine Sample Buffer (Bio-Rad) containing 2% BME. Whole cell lysates were heated to 100°C for 10 minutes and stored at -20°C. For tumor cell lines, cells were scraped into 1 mL media, centrifuged at 2000 x g, and washed with PBS. Pellets were resuspended in lysis buffer [20 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 1 mM DTT, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, Complete Protease Inhibitor Cocktail (Roche Applied Science), and Phosphatase Inhibitor Cocktail 3]. Protein concentration was determined using DC Protein Assay (Bio-Rad). Proteins (20 to 40 μg) were resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies overnight at 4°C. Primary antibody information can be found in S2 Table. AffiniPure HRP-conjugated goat anti-rabbit, donkey anti-goat, and goat anti-mouse IgG secondary antibodies were from Jackson ImmunoResearch.

Samples were mixed with an equal volume of tricine sample buffer (BioRad, Hercules CA) and then electrophoresed on 12.5% sodium dodecyl sulfate-tris-tricine polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes via electroblotting as previously described [27 (link)]. Membranes were stained with reversible stain Fast Green FCF 0.1% (Fisher Scientific, Waltham, MA) in 25% methanol-10% acetic acid for 1 min, destained with 25% methanol, and then transferred to distilled water to assess equal loading. Membranes were then washed in TBST and blocked for 1 h at room temperature with 5% nonfat dry milk in TBST. Blots were then incubated with PHF1 (1:500), 6E10/4G8 (1:2000), and actin (1:5000; Sigma) overnight at 4 °C. The next day, bound antibodies were detected after 1 h incubation with goat anti-mouse or anti-rabbit IgG HRP (1:3000; GE Healthcare, UK) and the chemiluminescent detection system (Pierce, Rockford, IL) on autoradiography films (MIDSCI, St. Louis, MO).

The SPA-tagged strains were grown in the respective growth medium used in ribosome profiling experiments, and harvested. Whole cells were resuspended in tricine sample buffer (Bio-Rad, Hercules, CA) and heated at 95° for 10 min. The total protein (equivalent to the number of cells at OD₆₀₀ 0.05) was separated on a 16.5% tricine gel (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad) according to the manufacturer’s protocol. The SPA-tagged protein was detected using a monoclonal anti-FLAG M2-alkaline phosphatase-conjugated antibody (Sigma-Aldrich, Saint Louis, MO) and CDP Star chemiluminescent substrate (Sigma-Aldrich) according to the manufacturer’s protocol. The Novex sharp prestained protein standard (Novex, Carlsbad, CA) was used as a size marker.

Protein samples from bacterial pellets and cell fractions were denatured in boiling water for 5 min in tricine sample buffer (BioRad). SDS-PAGE was used to separate proteins with either Any kD Tris-glycine precast gels or a 16.5% Tris-Tricine precast gels (BioRad) prior to western blotting. The Tris-Tricine gels were used to improve resolution for McpM. A Trans-Blot turbo transfer starter system (BioRad) was used to transfer proteins to a low-fluorescence polyvinylidene fluoride (LF-PVDF) membrane. Primary antibodies anti-His-tag (1:2500, Novagen), anti-DnaK (1:5000, Abcam) were used with secondary goat anti-mouse antibody (1:5000, DyLight 488 conjugate) to visualize proteins on western blots. A ChemiDoc MP Imaging System (BioRad) was used to detect fluorescent signal.

For electrophoresis to confirm the identity of aβComAb GW-23B7, 1 μg of antibody with or without dithiothreitol (DTT) 0.1 M was mixed with an equal volume of tricine sample buffer (Bio-Rad Laboratories, Hercules, CA, USA), electrophoresed on Bolt™ 4–12% Bis-Tris (Thermo Fisher Scientific) polyacrylamide gels and system, and transferred onto nitrocellulose membranes (NCs) for 1 h at 386 mA in 0.1% 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) (Sigma-Aldrich, St. Louis, MO, USA)-10% methanol. Equal protein loading was assessed by reversible protein stain Fast Green (FG) FCF 0.1% (Thermo Fisher Scientific) in 25% methanol-10% acetic acid for 1 minute, destained with 25% methanol, transferred to distilled water, and scanned on a Canon F916900 scanner (Canon Inc., Beijing, China). Membranes were then washed in TBS-T until the stain was eliminated, blocked 1 h at RT with 5% nonfat dry milk in TBS-T, pH 8.3, and incubated with HRP-conjugated rat antimouse IgM(μ) heavy chain-specific (1:6000; Thermo Fisher Scientific) or HRP-conjugated goat antimouse kappa (1:6000; SouthernBiotech). Bound antibodies were detected with an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology) on autoradiographic films (MIDSCI, St. Louis, MO, USA).