LB media with different pH were prepared by dissolving LB powder (DifcoTM LB Broth, BD) in PBS followed by pH adjustment, and then sterilized by filtering with 0.2 μM membranes. The sample preparation and western blotting of LTA were performed based on the previous studies of Gründling et al. 19 (link),22 (link). Briefly, S. aureus cells were cultured in LB at 37°C, collected and resuspended in 500 μl of TBS buffer (20 mM Tris-Cl, pH 7.4, 150 mM NaCl). Bacteria were lysed by bead beater in 4 runs of 30 seconds per cycle with 6 m/s. The cell lysate was separated on 12% SDS-PAGE followed by western blot analysis. LTA was detected with LTA (polyglycerolphosphate)-specific primary antibody (clone 55, HyCult Biotechnology) and antimouse IgG (H+L) HRP antibody (Promega) as the secondary antibody. The blots were developed with SuperSignalTM west pico plus chemiluminescent substrate (Thermo Fisher Scientific), and the images were obtained with a Fusion FX7 imaging system (Witec). The relative amount of LTA was determined from the band intensities of western blots (n=4) using ImageJ (https://imagej.nih.gov).
Fusion fx7 imaging system
Manufactured by WITec
The Fusion FX7 is a high-performance imaging system that combines advanced microscopy techniques to provide comprehensive analysis of samples. The system integrates multiple imaging modalities, enabling users to capture detailed information about the structure and composition of materials at the micro- and nanoscale.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ubch5a, by R&D Systems (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Supersignal west pico plus chemiluminescent substrate solution, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl solution, by Bio-Rad (1 mentions)
Nupage bis tris system, by Thermo Fisher Scientific (1 mentions)
3 protocols using fusion fx7 imaging system
1
Quantification of Staphylococcal Lipoteichoic Acid
2
In vitro Ubiquitination Assay for CRL4 E3 Ligase
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In vitro Ubiquitination was performed by mixing wild‐type or mutant CRL4DCAF1 at 2 μM with a reaction mixture containing E1 (UBA1, Boston Biochem) at 0.1 μM, E2 (UBCH5a, Boston Biochem) at 0.1 μM, wild‐type Ubiquitin (Ubiquitin, Boston Biochem) at 5 μM as indicated. Reactions were carried out in 50 mM Tris pH 7.7, 200 mM NaCl, 10 mM MgCl2, 0.2 mM CaCl2, 3 mM ATP, 2 mM DTT, and 0.1 mg/ml BSA, and incubated for 0–15 min at 30°C. Reactions were then analyzed by Western blot using anti‐Ubiquitin (P4D1) primary antibody (Santa Cruz, 1:500) and Horseradish Peroxidase (HRP) conjugated anti‐rabbit secondary antibody (1:10,000). Blots were incubated with SuperSignal™ West Pico PLUS Chemiluminescent Substrate solution (Thermo Fisher) and scanned on Fusion FX7 imaging system (Witec AG).
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In vitro Ubiquitination was performed by mixing wild‐type or mutant CRL4DCAF1 at 2 μM with a reaction mixture containing E1 (UBA1, Boston Biochem) at 0.1 μM, E2 (UBCH5a, Boston Biochem) at 0.1 μM, wild‐type Ubiquitin (Ubiquitin, Boston Biochem) at 5 μM as indicated. Reactions were carried out in 50 mM Tris pH 7.7, 200 mM NaCl, 10 mM MgCl2, 0.2 mM CaCl2, 3 mM ATP, 2 mM DTT, and 0.1 mg/ml BSA, and incubated for 0–15 min at 30°C. Reactions were then analyzed by Western blot using anti‐Ubiquitin (P4D1) primary antibody (Santa Cruz, 1:500) and Horseradish Peroxidase (HRP) conjugated anti‐rabbit secondary antibody (1:10,000). Blots were incubated with SuperSignal™ West Pico PLUS Chemiluminescent Substrate solution (Thermo Fisher) and scanned on Fusion FX7 imaging system (Witec AG).
3
Western Blot Sample Preparation and Imaging
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Samples were either run on 4–12% NuPAGE or Bolt gels with the NuPAGE Bis–Tris system (Invitrogen) in MOPS SDS buffer or on 8% gels with the Bio‐Rad system with SDS‐running buffer. Western blotting was performed with the wet transfer systems from Bio‐Rad on 0.45 μm Nitrocellulose membranes (Amersham™), which were blocked in 5% milk in PBS. Primary antibody dilutions were 1:500 in 5% milk‐PBS, while secondary antibodies were diluted 1:2,000. Blots were visualized with SuperSignal™ West Pico PLUS or Femto Chemiluminescent Substrate solution (Thermo Fischer) or with Clarity™ Western ECL solution (Bio‐Rad) and scanned on Fusion FX7 imaging system (Witec AG).
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