Cells were grown at 30 °C in M9-based medium with 20 μg/ml each of the 20 amino acids, 2 μg/ml thiamine, and 0.4% glucose until mid-log phase. Total cellular proteins were precipitated with 5% trichloroacetic acid, washed with acetone, and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (90 (link), 91 (link)). Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (MilliporeSigma). Only when 15% bis-Tris gel was used for SDS-PAGE, a transferred membrane filter was dried at 37 °C for 30 min and then hydrophilized with methanol. After blocking with BLOTTO (90 (link)), the filter was incubated with an appropriate antibody. For anti-RseP immunoblotting, anti-RseP antibodies were preincubated with whole-cell lysates of AD1840 (the ΔrseA ΔrseP ΔdegS strain) at 4 °C for 1 h to reduce a background as described previously (82 (link)). The filter was then washed and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxide (Bio-Rad). After washing of the filter, proteins that reacted with secondary antibodies were visualized using ECL or ECL Prime Western Blotting Detection Reagents (Cytiva) and Bio image analyzer LAS4000mini (Cytiva).
Bio image analyzer las4000mini
Manufactured by Cytiva
The Bio image analyzer LAS4000mini is a compact and versatile imaging system designed for a wide range of applications in life science research. It provides high-quality, sensitive detection and quantification of various fluorescent and chemiluminescent signals, including Western blots, gel documentation, and microarray analysis.
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2 protocols using bio image analyzer las4000mini
1
Immunoblot Analysis of RseP Protein
2
Immunoblotting Analysis of RseP Protease
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Cells were grown at 30°C in M9-based medium with 20 μg/mL each of the 20 amino acids, 2 μg/mL thiamine and 0.4% glucose until mid-log phase. Total cellular proteins were precipitated with 5% trichloroacetic acid (TCA), washed with acetone and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (90, 91) . Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (MilliporeSigma). Only when 15% bis-Tris gel was used for SDS-PAGE, a transferred membrane filter was dried at 37°C for 30 min and then hydrophilized with methanol. After blocking with BLOTTO (90), the filter was incubated with an appropriate antibody. For anti-RseP immunoblotting, anti-RseP antibodies were pre-incubated with whole-cell lysates of AD1840 (the ΔrseA ΔrseP ΔdegS strain) at 4°C for 1 h to reduce a background as described previously (83) . The filter was then washed, and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxide (Bio-Rad). After washing of the filter, proteins that reacted with secondary antibodies were visualized using ECL or ECL Prime Western Blotting Detection Reagents (Cytiva)
and Bio image analyzer LAS4000mini (Cytiva).
and Bio image analyzer LAS4000mini (Cytiva).
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