Cells were washed twice in ice-cold PBS and immediately lysed in Laemmli buffer as described previously [38 (link)]. Equal amounts of cell lysates were resolved by 10% SDS/PAGE and electro-transferred onto poly(vinylidene difluoride) membrane. Immunoblot analyses were performed as described previously [19 (link)] using the primary antibodies: mouse monoclonal anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-p73 (Ab-4; NeoMarkers, Fremont, CA, USA), mouse monoclonal anti-γH2AX (2F3; BioLegend, San Diego, CA, USA), rabbit polyclonal anti-p21WAF1 (Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-p53 at Ser-15 (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-RUNX2 (Cell Signaling Technology), rabbit polyclonal anti-E2F-1 (Cell Signaling Technology), rabbit polyclonal anti-PARP (Cell Signaling Technology), rabbit polyclonal anti-caspase-9 (Cell Signaling Technology) and rabbit polyclonal anti-Actin (20–33; Sigma, St Louis, MO, USA) antibodies. Immunoreactive bands were visualized by an enhanced chemiluminescence system (ECL; GE Healthcare, Little Chalfont, UK) in accordance with the manufacturer's instructions.
Protocol detail
Western Blot Analysis of Cellular Proteins
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
mouse monoclonal anti-p53 (DO-1;
anti-RUNX2
rabbit polyclonal anti-E2F-1
rabbit polyclonal anti-PARP
anti-caspase-9
anti-Actin
Actin
Antibodies
Caspase 9
Cell
Chemiluminescence
Cold
E2f 1
Immunoblot analyses
Laemmli buffer
Membrane
Mouse
Poly
Rabbit
Runx2
Sds page
Corresponding Organization : Chiba Cancer Center
Other organizations : Nihon University
Top 5 similar protocols
Variable analysis
independent variables
- None explicitly mentioned
- P53 protein levels
- P73 protein levels
- γH2AX protein levels
- P21WAF1 protein levels
- Phospho-p53 (Ser-15) protein levels
- RUNX2 protein levels
- E2F-1 protein levels
- PARP protein levels
- Caspase-9 protein levels
- Actin (used as a loading control)
- Positive control: None mentioned
- Negative control: None mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!