Western Blot Analysis of GFP-tagged Proteins

Preparation of protein extracts and Western blot analysis were performed as described previously [10 (link)]. WIDE RANGE Gel Preparation Buffer(4x) for PAGE (Nakalai) was used to detect degradation of GFP-tagged proteins. Anti-GFP monoclonal antibody JL-8 (Clontech), anti-GFP antibody from mouse IgG1κ (clones 7.1 and 13.1) (Roche), anti-phospho-AMPKα monoclonal antibody 40H9 (Cell Signaling), anti-Snf1 polyclonal antibody yk-16 (Santa Cruz), anti-Myc monoclonal antibody 9E10 (Santa Cruz) and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore) or the Odyssey Imaging Systems (LI-COR Biosciences). Signal intensities were quantified by the Odyssey Imaging Systems, and statistical analysis was performed with Excel (Microsoft).

Free full text: Click here

Mizuno T., Muroi K, & Irie K. (2020). Snf1 AMPK positively regulates ER-phagy via expression control of Atg39 autophagy receptor in yeast ER stress response. PLoS Genetics, 16(9), e1009053.

Publication 2020

Anti-GFP monoclonal antibody JL-8 anti-GFP antibody from mouse IgG1κ (clones 7.1 and 13.1) anti-phospho-AMPKα monoclonal antibody 40H9 anti-Snf1 polyclonal antibody yk-16 anti-Myc monoclonal antibody 9E10 anti-Mcm2 polyclonal antibody N-19 LAS-4000 Immobilon Western Odyssey Imaging Systems

Anti antibody Buffer Clones Gel preparation Immobilon Mcm2 Mouse Protein Proteins degradation Western blot analysis

Corresponding Organization : University of Tsukuba

Top 5 similar protocols

Variable analysis

independent variables

Preparation of protein extracts
Western blot analysis

dependent variables

Degradation of GFP-tagged proteins
Phosphorylation of AMPKα
Levels of Snf1, Myc, and Mcm2 proteins

control variables

WIDE RANGE Gel Preparation Buffer(4x) for PAGE (Nakalai) used to detect GFP-tagged protein degradation
Anti-GFP monoclonal antibody JL-8 (Clontech), anti-GFP antibody from mouse IgG1κ (clones 7.1 and 13.1) (Roche), anti-phospho-AMPKα monoclonal antibody 40H9 (Cell Signaling), anti-Snf1 polyclonal antibody yk-16 (Santa Cruz), anti-Myc monoclonal antibody 9E10 (Santa Cruz) and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) used for detection
Detection carried out using LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore) or the Odyssey Imaging Systems (LI-COR Biosciences)
Signal intensities quantified by the Odyssey Imaging Systems, and statistical analysis performed with Excel (Microsoft)

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!