Criterion tgx precast protein gel

Harvested cells were lysed in sodium dodecyl sulfate (SDS) sample buffer at 10⁶ cell/10ul. After sonication, protein lysates were denatured at 100°C for 5 min and resolved on 10% SDS-polyacrylamide gel or 4–15% Criterion TGX Precast Protein Gel (Bio-Rad). Resolved proteins were transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked in TBS buffer (50 mM Tris-Cl, pH 7.5 and 150 mM NaCl) supplemented with 5% non-fat milk and 0.1% Tween-20. nitrocellulose membranes were blotted with specific primary antibodies to cellular and viral proteins. Immunocomplexes were visualized by ECL (GE) or by autoradiography using ¹²⁵I-protein A (PerkinElmer).

Wild type, recipient, and transconjugant strains were grown in IR and ID LB broth overnight at 37 °C with shaking. Overnight cultures were diluted (1:20) in to corresponding fresh IR and ID LB media and cell lysates were prepared at mid-logarithmic phase. To determine the approximate bacterial cell numbers, optical density (OD) was measured at 600 nm, using a Genesys 10UV spectrophotometer (Thermo Electron Corp., Madison, WI). Bacterial cell pellets were dissolved in protein sample buffer (Bio-Rad, Hercules, CA), boiled at 100 °C for 10 min and 25 μL of cell lysates containing equal number of bacterial cells (~ 10⁸ cells) were loaded onto a 4 to 15% gradient Criterion TGX precast protein gel (Bio-Rad, Hercules, CA). Protein bands were separated on a Criterion electrophoresis chamber and visualized by staining with Coomassie blue. Triplicate protein gels were run and stained using protein lysates collected from three independent experiments in order to examine the consistent differential expressions of protein bands from each experiment. Differently expressed protein bands were selected from one representative gel, cut and sent to MS Bioworks (Ann Arbor, MI) for further proteome analyses.
At MS Bioworks, protein content in gel bands were identified by nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described earlier [45 (link)].

Samples were loaded onto a 4–20% Criterion TGX Precast Protein Gel (Bio-Rad) [7 (link)]. Protein was transferred onto a nitrocellulose membrane and probed for Fpn (Alpha Diagnostics, MTP11-S, 1:1000), DMT1 (Millipore, ABS983, 1:1000), Heph (Santa Cruz, SC-365365, 1:1000), TfR (Santa Cruz, sc-65882, 1:250), Tf (Abcam, ab82411, 1:1000), HA tag (Invitrogen, MA5-27915, 1:1000), ubiquitin (Protein Tech, 10201–2-AP, 1:1000) or cyclophilin B (Abcam, ab16045, 1:1000) as a loading control. Corresponding secondary antibody conjugated to HRP was used (1:5000, GE Amersham) and bands were visualized using ECL reagents (Perkin-Elmer) on an Amersham Imager 600 (GE Amersham). Cellular lysate samples were normalized to cyclophilin B protein as a loading control, and then subsequently normalized to an untreated control sample within each experiment. Membrane protein samples were stained with Ponceau S and normalized to total protein as a loading control.