Cells were harvested by centrifugation, washed once with PBS, and lysed in RIPA buffer (Cell Signaling) according to the manufacturer’s instructions. Protein concentration of the lysates was determined by the bicinchoninic acid (BCA) method according to the manufacturer’s recommendations (Thermo Scientific). About 10 to 20 μg of protein per sample was separated on 10% Mini-Protean TGX gels (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Life Technologies). Western blot analysis was performed according to standard protocols. Total PPM1A, α-tubulin, or GAPDH proteins were detected with specific antibodies (see antibody section). A horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibody (Santa Cruz) was used as the secondary antibody. The blot was developed using the Western Lightning Ultra chemiluminescent substrate from Perkin Elmer, Inc., and detected in an EpiChemi3 darkroom (UVP BioImaging Systems).
Western lightning ultra chemiluminescent substrate
Manufactured by PerkinElmer
The Western Lightning Ultra chemiluminescent substrate is a sensitive luminol-based reagent used to detect and quantify proteins in Western blot analyses. It generates a chemiluminescent signal in the presence of the target proteins, which can be captured and measured using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Iblot gel transfer system, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated goat anti rabbit or goat anti mouse polyclonal antibody, by Santa Cruz Biotechnology (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Fusion sl chemiluminescent imager, by Avantor (1 mentions)
4 protocols using western lightning ultra chemiluminescent substrate
1
Western Blot Analysis of PPM1A Protein
2
Western Blot Analysis of PPM1A Protein
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Cells were harvested by centrifugation, washed once with PBS, and lysed in RIPA buffer (Cell Signaling) according to the manufacturer’s instructions. Protein concentration of the lysates was determined by the bicinchoninic acid (BCA) method according to the manufacturer’s recommendations (Thermo Scientific). About 10 to 20 μg of protein per sample was separated on 10% Mini-Protean TGX gels (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Life Technologies). Western blot analysis was performed according to standard protocols. Total PPM1A, α-tubulin, or GAPDH proteins were detected with specific antibodies (see antibody section). A horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibody (Santa Cruz) was used as the secondary antibody. The blot was developed using the Western Lightning Ultra chemiluminescent substrate from Perkin Elmer, Inc., and detected in an EpiChemi3 darkroom (UVP BioImaging Systems).
3
Protein Quantification and Western Blot Analysis
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Cells were harvested by centrifugation, washed once with PBS, and lysed in RIPA buffer (Cell Signaling) according to the manufacturer's instructions. Protein concentration of the lysates was determined by the bicinchoninic acid (BCA) method according to the manufacturer's recommendations (Thermo Scientific). About 10 to 20 μg of protein per sample was separated on 10% Mini-Protean TGX gels (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Life Technologies). Western blot analysis was performed according to standard protocols. Total PPM1A, α-tubulin, or GAPDH proteins were detected with specific antibodies (see antibody section). A horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibody (Santa Cruz) was used as the secondary antibody. The blot was developed using the Western Lightning Ultra chemiluminescent substrate from Perkin Elmer, Inc., and detected in an EpiChemi3 darkroom (UVP BioImaging Systems).
4
FLAG-MetK Protein Expression Analysis
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Production of the fusion FLAG-MetK protein was tested by Western blot analysis. Cells were harvested by centrifugation at 4°C and resuspended in a sample buffer [62 ]. Before loading on a gel, samples were heated to 95°C for 10 min. Samples corresponding to an OD600 nm of 1 were separated in 10% polyacrylamide gels by SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using a Semi-Dry Blotter. For protein detection, monoclonal, horseradish peroxidase-conjugated antibody raised against the FLAG tag (Sigma Aldrich) and Western Lightning Ultra, Chemiluminescent Substrate (PerkinElmer) were used. To visualize the signals, a Fusion-SL chemiluminescent imager (Peqlab) was used.
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