Proteins were separated by SDS–PAGE under reducing conditions and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% tween 20 (PBST). The blots were incubated overnight at 4°C with primary antibodies. Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare).
Enhanced chemiluminescence procedure
Manufactured by GE Healthcare
Enhanced chemiluminescence procedure is a laboratory technique used to detect and quantify proteins in a sample. It involves the use of a chemiluminescent substrate, which emits light when it reacts with the target protein. The intensity of the emitted light is proportional to the amount of the target protein present, allowing for its detection and quantification.
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4 protocols using enhanced chemiluminescence procedure
1
Protein Detection by Immunoblotting
2
Western Blot Analysis of Protein Expression
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For Western blot analyses, the indicated amounts of cell extracts were resolved in 10% SDS–PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, BioScience, Dassel, Germany). Membranes were blocked with 5% (wt/vol) nonfat dry milk in phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and probed with monoclonal anti-FLAG antibody (Sigma), monoclonal anti-APE1 antibody (Vascotto et al., 2009a (link)), monoclonal anti-Ku70 (sc-12729; Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti–RNA polymerase II (Abcam, Cambridge, MA), monoclonal anti-SIRT1 (Abcam), monoclonal anti-p32 (Santa Cruz Biotechnology), and polyclonal anti-p53(acetyl K382) (Abcam). Blots were developed by using the enhanced chemiluminescence procedure (GE Healthcare, Piscataway, NJ) or Western Lightning Ultra (Perkin Elmer, Waltham, MA). Data normalization was performed by using a monoclonal anti-tubulin antibody (Sigma). Blots were quantified by using a Chemidoc XRS video densitometer (Bio-Rad, Hercules, CA).
3
Western Blot Analysis of Proteins
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Proteins were separated by SDS–PAGE under reducing conditions and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% tween 20 (PBST). The blots were incubated overnight at 4°C with primary antibodies. Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare).
4
Protein Expression Analysis
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Cells subjected to the treatment indicated in the appropriate figure legend were harvested, lysed, and protein concentration determined according to standard protocol. Proteins were separated by SDS-PAGE, and immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare, Piscataway, NJ).
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