Western Blot Analysis of Protein Expression

Total protein was extracted with RIPA buffer (Pierce, no. 89900) supplemented with protease inhibitor (Life Technologies, no. 1861280) and quantified using the BCA Protein Assay (ThermoFisher Scientific). Proteins were separated on Bolt 4-12% Bis-Tris Plus gels (Life Technologies, no. NW04120) and transferred to polyvinylidene difluoride membranes. Membranes were probed with antibodies against GCLC (Proteintech, no. 12601-1-AP, 1:2000 dilution), HuR (Santa Cruz Biotechnology, no. SC-5261, clone 3A2, 1:2000 dilution)^{21 (link),55 (link)–60 (link)}, Lamin A/C (Cell Signaling, no. 4777, 1:2000 dilution), α-Tubulin (Proteintech, no. 11224-1-AP, 1:4000 dilution), and β-Actin (Santa Cruz Biotechnology, no. SC-47778, 1:4000 dilution). Blots were probed with secondary antibodies customized for the Odyssey Imaging system Secondary antibodies (680RD Goat anti-Mouse IgG (Li-COR, no. 926-68070, 1:20000 dilution) and 800CW Goat anti-Rabbit IgG (Li-COR, no. 926-32211, 1:10000 dilution). The density of blots was quantified using Image Studio Software v.5.2.5.

Free full text: Click here

Vaziri-Gohar A., Hue J.J., Abbas A., Graor H.J., Hajihassani O., Zarei M., Titomihelakis G., Feczko J., Rathore M., Chelstowska S., Loftus A.W., Wang R., Zarei M., Goudarzi M., Zhang R., Willard B., Zhang L., Kresak A., Willis J.E., Wang G.M., Tatsuoka C., Salvino J.M., Bederman I., Brunengraber H., Lyssiotis C.A., Brody J.R, & Winter J.M. (2023). Increased glucose availability sensitizes pancreatic cancer to chemotherapy. Nature Communications, 14, 3823.

Publication 2023

protease inhibitor BCA Protein Assay Bolt 4-12% Bis-Tris Plus gels Lamin A/C α-Tubulin β-Actin 680RD Goat anti-Mouse IgG 800CW Goat anti-Rabbit IgG

Actin Anti igg Antibodies Assay Bis tris Buffer Clone Dilution Gclc Gels Goat Lamin a c Membranes Mouse Polyvinylidene difluoride Protease inhibitor Proteins Rabbit Ripa α tubulin

Corresponding Organization : Case Western Reserve University

Other organizations : University Hospitals of Cleveland, Thomas Jefferson University, Texas A&M University, Cleveland Clinic, University of Pittsburgh, The Wistar Institute, Michigan Medicine, Oregon Health & Science University

Top 5 similar protocols

Variable analysis

independent variables

Extraction buffer (RIPA buffer)
Protease inhibitor
Antibodies (GCLC, HuR, Lamin A/C, α-Tubulin, β-Actin)

dependent variables

Total protein quantity (measured by BCA Protein Assay)
Protein expression levels (measured by Western blot)

control variables

Gel type (Bolt 4-12% Bis-Tris Plus gels)
Membrane type (polyvinylidene difluoride membranes)
Secondary antibodies (680RD Goat anti-Mouse IgG, 800CW Goat anti-Rabbit IgG)
Imaging system (Odyssey Imaging system)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly 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!