Whole proteins were extracted in RIPA buffer (Cell Signaling) and quantified by the Bradford protein assay (Biorad). Samples were separated by 12% SDS–PAGE and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare). The membranes were blocked with Blotto A (Santa Cruz) at room temperature for 1 h, and incubated with the appropriate primary antibodies for 2 h at room temperature, as previously described (Vergara et al., 2016 (link)). After two washes with a solution of TBS containing 0.1% (v/v) tween 20 for 10 min, the membranes were incubated with secondary antibody HRP-conjugated for 2 h at room temperature (standard dilution 1:2,000). Blots were then developed using the Amersham ECL western blotting detection system (GE Healthcare). Densitometric quantitation of at least three independent replicates was done using ImageJ software.
Blotto a
Manufactured by Santa Cruz Biotechnology
Blotto A is a laboratory buffer solution used in western blotting and other immunodetection techniques. It serves as a blocking agent to prevent non-specific binding of antibodies to the membrane, improving the specificity of the target protein detection.
Automatically generated - may contain errors
3 protocols using blotto a
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole proteins were extracted with RIPA buffer (Cell Signaling) and quantified by the Bradford protein assay (Bio-Rad). Samples were separated by 10% SDS-PAGE and transferred to the Hybond ECL nitrocellulose membrane. The membranes were blocked overnight in Blotto A (Santa Cruz) at 4°C and subsequently probed by the appropriately diluted primary antibodies for 2 h at room temperature. Protein bands were visualized by incubating with a horseradish peroxidase-conjugated secondary antibody (Amersham, ECL Western blotting detection reagents).
3
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole proteins were extracted in RIPA buffer (Cell Signaling, Danvers, MA) and quantified by the Bradford protein assay (BIORAD, Hercules, CA). Samples were separated by 12% SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The membranes were blocked with Blotto A (Santa Cruz, Dallas, Texas) at room temperature for 1 h, and incubated with the appropriate primary antibodies (see Supplementary Table 1) for 2 h at room temperature, as previously performed. 20 After two washes with a solution of TBS containing 0.1% (v/v) tween 20 for 10 min, the membranes were incubated with secondary antibody HRPconjugated for 2 h at room temperature (standard dilution 1:2000). Blots were then developed using the Amersham ECL western blotting detection system (GE Healthcare, Little Chalfont, UK).
Densitometric quantitation of at least three indipendent replicates was done using ImageJ software.
Densitometric quantitation of at least three indipendent replicates was done using ImageJ software.
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