Proteins were separated by 10-12% SDS-PAGE and transferred onto PVDF membrane (Immobilon-P membrane; Millipore; Bedford, MA, USA). Membranes were incubated with specific antibodies, followed by washing and incubation with a horseradish peroxidase-conjugated secondary antibody. Specific antibody-antigen complex was detected using an enhanced chemiluminescence Western Blot detection system (Thermo Fisher Scientific Inc.). The amount of detected proteins was quantified by ImageJ software (NIH, Bethesda, MD, USA), and was expressed as the ratio to β-actin protein. Each assay was performed in quadruplicates.
Enhanced chemiluminescence western blot detection system
Manufactured by Thermo Fisher Scientific
The Enhanced chemiluminescence Western Blot detection system is a laboratory equipment used to visualize and quantify specific proteins in a sample. It utilizes a chemiluminescent substrate to generate a light signal that is proportional to the amount of target protein present. The system is designed to provide sensitive and accurate detection of proteins in Western blot analysis.
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Lab products found in correlation
3 protocols using enhanced chemiluminescence western blot detection system
1
Quantitative Protein Analysis by Western Blot
2
Quantitative Analysis of NIS and MUC20 Proteins
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Different groups of WRO cells were seeded into 24-well plates with a cover glass over each well and cultured for 24 h. Total, membranous and cytoplasmic NIS and MUC20 proteins were extracted according to the standard steps of the protein extraction kits.
Lysates were quantified spectrophotometrically using the bicinchoninic-acid-based (BCA) method. Twenty-five micrograms of each sample was separated on gradient polyacrylamide gels and transferred onto polyvinyldifluoride membranes. Membranes were incubated with a primary antibody (anti-NIS, Bioss, China; MUC 20, Abnova, USA; MET, abcam, UK; MET pY1234/5, CST, USA; β‐actin, CST, USA) overnight at 4°C in TTBS/milk. The samples were subsequently incubated with horseradish peroxidase-conjugated secondary anti-mouse antibodies (CST, USA). The protein was visualized using an enhanced chemiluminescence Western blot detection system (Thermo Fisher Scientific). Multiplication of the intensity and area of protein bands indicated the relative levels of protein expression.
Lysates were quantified spectrophotometrically using the bicinchoninic-acid-based (BCA) method. Twenty-five micrograms of each sample was separated on gradient polyacrylamide gels and transferred onto polyvinyldifluoride membranes. Membranes were incubated with a primary antibody (anti-NIS, Bioss, China; MUC 20, Abnova, USA; MET, abcam, UK; MET pY1234/5, CST, USA; β‐actin, CST, USA) overnight at 4°C in TTBS/milk. The samples were subsequently incubated with horseradish peroxidase-conjugated secondary anti-mouse antibodies (CST, USA). The protein was visualized using an enhanced chemiluminescence Western blot detection system (Thermo Fisher Scientific). Multiplication of the intensity and area of protein bands indicated the relative levels of protein expression.
3
Hippocampal Protein Expression Analysis
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The indicated protein expression in hippocampal tissue was measured by western blot analysis as described previously (Chen et al., 2019) (link). Briefly, hippocampal tissues were homogenized in 300 µL of radioimmunoprecipitation assay buffer. Thirty micrograms of total protein was loaded onto each lane, and the proteins were separated by SDS-PAGE in 10% polyacrylamide and electrotransferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 at room temperature for 1 h and then incubated with the appropriate primary antibody (BDNF, phosphorylated ERK1/2, ERK1/2, phosphorylated TrkB, TrkB, and β-actin; Abcam) for 2 h. After washing, each protein was identified with an enhanced chemiluminescence western blot detection system (Thermo Fisher Scientific Inc.) using an appropriate horseradish peroxidaseconjugated secondary antibody for 1 h. Detection and quantification of protein levels in the western blot were analyzed using an LAS-4000 chemiluminescence detection device (Fujifilm; Huang et al., 2019) (link).
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