Cells were washed twice with ice-cold PBS and lysed using standard RIPA buffer containing proteinase inhibitors (Beyotime, Jiangsu, China). Following quantification, protein samples were heated to 95°C for 5 minutes, then separated in a SDS-polyacrylamide gel and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk in TBS buffer for 1 hour and then incubated with primary antibodies overnight at 4°C. After washing with TBST buffer for 4*10 min, the membranes were incubated with HRP-conjugated secondary antibody for 2 hours at room temperature. The immune reaction was visualized using ECL Plus substrates (Roche, Basel, Switzerland).
Ecl plus substrates
Manufactured by Roche
Sourced in Switzerland
ECL Plus substrates are chemiluminescent reagents used for the detection of proteins in Western blot analysis. These substrates generate a luminescent signal upon reaction with the enzyme horseradish peroxidase (HRP), which is commonly used as a reporter in immunoassays. The intensity of the luminescent signal is proportional to the amount of target protein present in the sample.
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Lab products found in correlation
2 protocols using ecl plus substrates
1
Western Blot Protein Analysis Protocol
2
Protein Extraction and Western Blot
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Cells were washed twice with ice-cold PBS and lysed using 120–200 μl standard RIPA buffer containing protein inhibitors (Beyotime, Jiangsu, China). Protein in loading buffer was denatured by heating at 100°C for 10 minutes and then separated on a SDS-polyacrylamide gel by electrophoresis. 40–60 μg of protein per lane was loaded. The proteins were transferred onto a PVDF membrane (Bio-Rad, CA). The membranes were incubated in primary antibodies overnight at 4°C and then incubated in HRP-conjugated secondary antibodies for 2 hours at room temperature. The signals were visualized with ECL Plus substrates (Roche, Basel, Switzerland).
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