Western Blotting of Cell Signaling Proteins

Western blotting was carried out as described in (31 (link)). Antibodies used were MDM2 (Ab-1) 1:300 (#: OP46-100UG, Merck Millipore), MDMX (#: A300287A-2 Bethyl laboratories), WIP1 (F-10) 1:200 (#: sc-376257, Santa Cruz Biotechnology), p53 1:500 (#: NCL-L-p53-DO7, Leica Microsystems Ltd.), phospho-p53^Ser-15 1:1000 (#: 9284 Cell Signalling), p21^WAF1 1:100 (#: OP64, Calbiochem), BAX 1:1000 (#: 2772S, Cell Signalling), cleaved caspase-3 1:1000 (#: 9664S, New England Biolabs Ltd.), actin 1:3000 (#: A4700, Sigma-Aldrich). Secondary goat anti-mouse/rabbit HRP-conjugated antibodies (#: P0447/P0448, Dako) were used at 1:1000. All antibodies were diluted in 5% milk/1 × TBS-tween (w/v). Proteins were visualised using enhanced chemiluminescence (GE Life Sciences) and X-ray film (Fujifilm). Densitometry was carried out using ImageJ software.

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.

Esfandiari A., Hawthorne T.A., Nakjang S, & Lunec J. (2016). Chemical inhibition of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) by GSK2830371 potentiates the sensitivity to MDM2 inhibitors in a p53-dependent manner. Molecular cancer therapeutics, 15(3), 379-391.

Publication 2016

OP46-100UG NCL-L-p53-DO7 phospho-p53Ser-15 BAX cleaved caspase-3 enhanced chemiluminescence X-ray film

Actin Anti antibodies Antibodies Caspase 1 Chemiluminescence Densitometry Goat Mdm2 Milk Mouse Proteins Rabbit Tween X ray film

Corresponding Organization : Newcastle University

Top 5 similar protocols

Variable analysis

independent variables

Antibodies used for Western blotting

dependent variables

Protein expression levels of MDM2, MDMX, WIP1, p53, phospho-p53^Ser-15, p21^WAF1, BAX, cleaved caspase-3, actin

control variables

Western blotting procedure as described in reference 31
Dilution factors for all antibodies
Detection method (enhanced chemiluminescence and X-ray film)
Densitometry analysis using ImageJ software

controls

Positive control: No explicit positive control mentioned
Negative control: No explicit negative control 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.

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?

Revolutionizing how scientists
search and build protocols!