Cells were lysed in RIPA lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitors (Sigma). Protein (approximately 5–10 µg) was loaded on precast 7.5% TGX gels (BioRad, Hercules, CA), blotting was performed as described previously [53 (link)] using polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Pittsburg, PA). Membranes were blocked, incubated overnight at 4°C with primary antibody (EZH2, p21, p53, phospho-p53 (S15), β-tubulin, Chk1, phospho-Chk1 (S317), Iκ-Bα, phospho-H2A.X, or Lamin-B (See Supplementary Table S2 ), washed, and then incubated with HRP-conjugated secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and protein signals were observed using a chemiluminescence system (Thermo Scientific, Schaumberg, IL), according to instructions provided by the manufacturer.
Hrp conjugated secondary antibody
Manufactured by LGC
The HRP-conjugated secondary antibody is a laboratory reagent used to detect and quantify target proteins in various immunoassay techniques. It consists of a secondary antibody that is chemically linked to the enzyme horseradish peroxidase (HRP). The HRP enzyme can catalyze a color-producing reaction, allowing for the visualization and measurement of the target protein.
Automatically generated - may contain errors
2 protocols using hrp conjugated secondary antibody
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were lysed in RIPA lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitors (Sigma). Protein (approximately 5–10 µg) was loaded on precast 7.5% TGX gels (BioRad, Hercules, CA), blotting was performed as described previously [53 (link)] using polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Pittsburg, PA). Membranes were blocked, incubated overnight at 4°C with primary antibody (EZH2, p21, p53, phospho-p53 (S15), β-tubulin, Chk1, phospho-Chk1 (S317), Iκ-Bα, phospho-H2A.X, or Lamin-B (See Supplementary Table S2 ), washed, and then incubated with HRP-conjugated secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and protein signals were observed using a chemiluminescence system (Thermo Scientific, Schaumberg, IL), according to instructions provided by the manufacturer.
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