Cell lysates were prepared in Laemmli buffer (Boston BioProducts) containing Benzonase Nuclease (Sigma, E8263), Complete EDTA-free proteinase inhibitor cocktail (Roche), and PhosStop phosphatase inhibitor cocktail (Roche) and fractionated by 4%–15% SDS-PAGE. For detection of phosphorylated ADAR1 proteins, 6 % SDS-polyacrylamide gel containing 25 μM of Phos-tag acrylamide (Wako) and 125 μM of MnCl2 was used. For confirmation of phosphorylation, cell lysates were treated with 4 units/μl of Lambda phosphatase (NEB) at 30°C for 30 min prior to Phos-tag immunoblotting analysis. Proteins were transferred to Immobilon-P nylon membrane (Millipore). Blots were blocked with 1% Blocker BSA buffer (ThermoFisher Scientific) and incubated with primary antibodies (Supplementary Table 7 ) overnight at 4°C. After the incubation with the secondary antibodies, membranes were developed with ECL (GE Healthcare).
Blocker bsa buffer
Manufactured by Thermo Fisher Scientific
Blocker BSA buffer is a protein-based solution used to block non-specific binding sites in various immunoassay and molecular biology applications. It helps to reduce background signal and improve the specificity of target analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop phosphatase inhibitor cocktail, by Roche (3 mentions)
Benzonase nuclease, by Merck Group (3 mentions)
Immobilon p nylon membrane, by Merck Group (3 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (3 mentions)
Complete edta free proteinase inhibitor cocktail, by Roche (3 mentions)
Laemmli buffer, by Boston BioProducts (2 mentions)
Phos tag acrylamide, by Fujifilm (2 mentions)
Lambda phosphatase, by New England Biolabs (2 mentions)
Blocker bsa bovine serum albumin buffer, by Thermo Fisher Scientific (1 mentions)
3 protocols using blocker bsa buffer
1
SDS-PAGE Analysis of Phosphorylated ADAR1
2
SDS-PAGE Analysis of Phosphorylated ADAR1
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Cell lysates were prepared in Laemmli buffer (Boston BioProducts) containing Benzonase Nuclease (Sigma, E8263), Complete EDTA-free proteinase inhibitor cocktail (Roche), and PhosStop phosphatase inhibitor cocktail (Roche) and fractionated by 4%–15% SDS-PAGE. For detection of phosphorylated ADAR1 proteins, 6 % SDS-polyacrylamide gel containing 25 μM of Phos-tag acrylamide (Wako) and 125 μM of MnCl2 was used. For confirmation of phosphorylation, cell lysates were treated with 4 units/μl of Lambda phosphatase (NEB) at 30°C for 30 min prior to Phos-tag immunoblotting analysis. Proteins were transferred to Immobilon-P nylon membrane (Millipore). Blots were blocked with 1% Blocker BSA buffer (ThermoFisher Scientific) and incubated with primary antibodies (Supplementary Table 7 ) overnight at 4°C. After the incubation with the secondary antibodies, membranes were developed with ECL (GE Healthcare).
3
Western Blot Analysis of Proteins
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Cell lysates were prepared in Laemmli buffer containing benzonase nuclease (Sigma), complete EDTA-free proteinase inhibitor cocktail (Roche), and PhosStop phosphatase inhibitor cocktail (Roche) and size-fractionated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted to Immobilon-P nylon membrane (Millipore). Membranes were blocked with 10% Blocker BSA (bovine serum albumin) buffer (Thermo Fisher Scientific) and incubated with the primary antibodies overnight at 4 °C. After incubation with each appropriate secondary antibody, bands were detected with ECL (GE Healthcare) using X-ray films. Antibodies were diluted in 10% Blocker BSA buffer (Thermo Fisher Scientific). Antibodies used in this study are listed in Supplementary Data 4 .
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