Cell lysates were prepared as described before with modifications (56 (link)). Specifically, cells were grown to OD600 = 0.4 in medium A and treated with 600 mM NaCl. Samples were collected at different time points by pelleting equal amounts of cells and flash freezing pellets in liquid nitrogen. For the immunoblot analysis, cell pellets were resuspended in 55 μL 1× BugBuster protein extraction reagent (Novagen) supplemented with 0.1% lysonase (Novagen) and lysed by pipetting up and down for 10 s. The lysates were heated at 95 °C for 5 min after the addition of 18 μL 4× loading buffer and equal amount of lysates were loaded on a 10% SDS polyacrylamide gel. Proteins were separated by gel electrophoresis and transferred to a nitrocellulose membrane. Sigma factor RpoS was detected with specific primary antibody (BioLegend) and horseradish peroxidase-conjugated secondary antibody (Sigma). Protein bands were detected by chemiluminescence using an ADVANCED imager (Intas).
Advanced imager
Manufactured by Intas
The ADVANCED imager is a high-performance laboratory equipment designed for imaging and analysis. It provides accurate and efficient image capture and processing capabilities for various applications.
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Lab products found in correlation
2 protocols using advanced imager
1
Quantification of RpoS Protein Levels
2
Western Blot Protein Detection
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Western blot samples were eluted and separated by SDS-PAGE as described above, then blotted to nitrocellulose membranes using standard procedures. After blocking the membrane in 5% (w/v) fat-free milk powder in TBS-T (TBS with 0.5% Tween-20), membranes were incubated with primary antibodies overnight at 4 °C. Membranes were then incubated with secondary HRP-coupled antibodies for 1 hr at room temperature, and visualized using an Intas Advanced Imager. Primary antibodies used were rabbit polyclonal anti-LAT (Upstate (Millipore)), mouse monoclonal anti-CD229/SLAMF3 (R&D Systems), rabbit polyclonal anti-PSMF1 (gift from Prof. Kreuger), and rabbit monoclonal anti-ZDHHC18 (Abcam).
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