Aliquots of 5 × 107 cells were harvested by centrifugation. The pellets were lysed in 0.2 M NaOH for 10 min on ice, and proteins were precipitated by the addition of 50 µL of 50% trichloroacetic acid. The samples were centrifuged at 16,100g for 10 min at 4°C, and the pellets were resuspended in 4× Laemmli buffer and heated for 5 min at 95°C. Samples were separated in a denaturing 7.5% 37.5:1 polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare). The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 primary antibody (Abcam, EL7.E1) that recognizes both the unphosphorylated and phosphorylated forms of Rad53. Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare).
El7 e1
Manufactured by Abcam
The EL7.E1 is a laboratory equipment product. It serves the core function of measuring and analyzing specific parameters or characteristics. Further details on its intended use are not available.
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Lab products found in correlation
2 protocols using el7 e1
1
Western Blot Analysis of Rad53 Phosphorylation
2
Rad53 Phosphorylation Analysis by Western Blot
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Aliquots of 5 × 10 7 cells were harvested by centrifugation. The pellets were lysed in 0.2 M NaOH on ice for 10 min and proteins were precipitated by the addition of 50 µL of 50% trichloroacetic acid. The samples were centrifuged at 16,100 g for 10 min at 4°C and the pellets were resuspended in 4× Laemmli buffer and heated for 5 min at 95°C. Samples were separated in a denaturing 7.5% 37.5:1 polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare).
The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 primary antibody (EL7.E1, Abcam), which recognizes both the unphosphorylated and phosphorylated forms of Rad53, anti-Cdc5 (11H12 and 4F10, Medimabs), anti-HA primary antibody (3F10, Roche), and anti-Pgk1 primary antibody (22C5D8, Abcam). Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare). All western blots were independently replicated in the laboratory.
The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 primary antibody (EL7.E1, Abcam), which recognizes both the unphosphorylated and phosphorylated forms of Rad53, anti-Cdc5 (11H12 and 4F10, Medimabs), anti-HA primary antibody (3F10, Roche), and anti-Pgk1 primary antibody (22C5D8, Abcam). Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare). All western blots were independently replicated in the laboratory.
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