GST-CIB1 Pulldown Assay Protocol

GST-CIB1 expressed in Escherichia coli was purified with the use of glutathione-conjugated agarose beads (Sigma). ASK1 variants were translated in vitro in the presence of [³⁵S]methionine with the use of a TNT reticulocyte lysate system (Promega). The ³⁵S-labled proteins were incubated at 4 °C for 4 h in a binding buffer^{54 (link)} with GST-fused proteins immobilized on glutathione-agarose beads. The bound ³⁵S-labeled proteins were eluted from the beads and analyzed by SDS-PAGE and with a Fuji BAS 2500 phosphoimager.

Free full text: Click here

Yoon K.W., Yang H.S., Kim Y.M., Kim Y., Kang S., Sun W., Naik U.P., Parise L.V, & Choi E.J. (2017). CIB1 protects against MPTP-induced neurotoxicity through inhibiting ASK1. Scientific Reports, 7, 12178.

Publication 2017

glutathione-conjugated agarose beads TNT reticulocyte lysate system BAS 2500 phosphoimager

Agarose Ask1 Cib1 Escherichia coli Glutathione Methionine Promega Proteins Reticulocyte Sds page

Corresponding Organization :

Other organizations : Korea University, Hoseo University, Thomas Jefferson University, University of North Carolina at Chapel Hill

Top 5 similar protocols

Variable analysis

independent variables

GST-CIB1 expression in Escherichia coli
ASK1 variant translation in vitro in the presence of [35S]methionine

dependent variables

Binding of 35S-labeled ASK1 variants to GST-fused proteins

control variables

Binding buffer used for incubation at 4 °C for 4 h
GST-fused proteins immobilized on glutathione-agarose beads

controls

Positive control: GST-fused proteins immobilized on glutathione-agarose beads
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!