Preparation of Isotope-Labeled spPH Domain

The ¹⁵N- or ¹³C/¹⁵N-labeled spPH domain (residues 1-108) was prepared by a previously described method [28 (link)]. In brief, the spPH domain was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21-Gold (DE3) (Agilent Technologies). The lysed supernatant was loaded on to a cOmplete His-Tag purification resin column (Roche), and the eluate was digested with thrombin (Cytiva) to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).

Free full text: Click here

Okuda M, & Nishimura Y. (2023). Structural polymorphism of the PH domain in TFIIH. Bioscience Reports, 43(7), BSR20230846.

Publication 2023

pET15b vector Escherichia coli BL21-Gold (DE3) thrombin Amicon Ultra device Superdex75 column

Device Escherichia coli Gold Hexa Histidine Resin Thrombin Vector

Corresponding Organization :

Other organizations : Yokohama City University, Hiroshima University

Top 5 similar protocols

Variable analysis

independent variables

Labeling of the spPH domain with ¹⁵N or ¹³C/¹⁵N

dependent variables

Not explicitly mentioned

control variables

Expression of the spPH domain as a hexa-histidine-tagged product in a pET15b vector in Escherichia coli BL21-Gold (DE3)
Purification of the spPH domain using a cOmplete His-Tag purification resin column, thrombin digestion to remove the histidine tag, and Superdex75 column chromatography

controls

Positive control: Expression and purification of the unlabeled spPH domain using the same method (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!