Total protein extracts were obtained with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) and then quantified by a bicinchoninic acid (BCA) kit (Beyotime). The proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel were transferred onto polyvinylidene fluoride (PVDF) membranes, followed by being impeded in 5% skimmed milk at room temperature for 2 h. Subsequently, the membranes were incubated with primary antibodies against ENAH (Abcam, 1:1000, ab124685), Ki67 (Abcam, 1:5000, ab254123), proliferating cell nuclear antigen (PCNA) (Abcam, 1:1000, ab92552), SF3B4 (Abcam, 1:2000, ab183483), matrix metallopeptidase 2 (MMP2) (Abcam, 1:1000, ab92536), matrix metallopeptidase 9 (MMP9) (Abcam, 1:1000, ab76003), Notch1 (Abcam, 1:1000, ab52627), hes family bHLH transcription factor 1 (HES1) (Abcam, 1:1000, ab108937), mastermind like transcriptional coactivator 1 (MAML1) (Abcam, 1:1000, ab155786) and GAPDH (Abcam, 1:1000, ab8245) overnight at 4°C and further incubated with HRP-conjugated secondary antibody (Abcam, 1:1000; ab7090) at room temperature for 2 h. The resulting bands were visualized under enhanced chemiluminescence (MilliporeSigma). Protein levels were quantified using Image J software (version 1.46; National Institutes of Health).
Ab124685
Manufactured by Abcam
Ab124685 is a recombinant monoclonal antibody against the protein of interest. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab7090, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab108937, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab52627, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
2 protocols using ab124685
1
Western Blot Analysis of Key Proteins
2
Protein Extraction and Western Blot Analysis
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Proteins were extracted using radio immunoprecipitation assay lysis buffer (Beyotime Biotechnology). Dodecyl sulfonate polyacrylamide gel electrophoresis Kit (Beijing Dingguo Changsheng Biotechnology) and the polyvinylidene fluoride (PVDF) membranes (Merck) were used to separate the protein, and PVDF membranes were closed with 5% non‐fat powdered milk (Solarbio) for 2 h. The primary antibodies to Bcl2‐associated X (Bax) (ab32503, 1:1000; Abcam), cyclin D1 (2978, 1:1000; Cell Signaling Technology), and ENAH (ab124685, 1:1000; Abcam) and immunoglobulin G secondary antibody conjugated with horseradish peroxidase (HRP) (ab201718, 1:5000; Abcam) were incubated with the membranes. The membranes were added with Immobilon Western Chemiluminescent HRP Substrate (Merck) and photographed in a gel imaging analyzer (UVITEC).
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