Purification of Recombinant Talin and Vinculin Proteins

Talin constructs were synthesized by PCR using a mouse talin1 cDNA as template, and cloned into pET-151/D-TOPO (Invitrogen). Constructs were expressed in Escherichia coli BL21 Star (DE3) cultured in LB. Recombinant His-tagged talin polypeptides were purified by nickel-affinity chromatography following standard procedures63 (link). The His-tag was removed by cleavage with AcTEV protease (Invitrogen), and the protein was further purified by anion-exchange chromatography. Concentration was determined using extinction coefficients at 280 nm. Recombinant His- tagged chicken vinculin domain 1 (Vd1; residues 1–258) was expressed from a pET-15b plasmid and purified via nickel-affinity chromatography followed by anion-exchange chromatography, as described by20 (link).

Free full text: Click here

Atherton P., Stutchbury B., Wang D.Y., Jethwa D., Tsang R., Meiler-Rodriguez E., Wang P., Bate N., Zent R., Barsukov I.L., Goult B.T., Critchley D.R, & Ballestrem C. (2015). Vinculin controls talin engagement with the actomyosin machinery. Nature Communications, 6, 10038.

Publication 2015

pET-151/D-TOPO AcTEV protease

Affinity chromatography Anion Cdna Chicken Chromatography Cleavage Escherichia coli Extinction Mouse Nickel Plasmid Polypeptides Protease Protein Talin Topo Vinculin

Corresponding Organization : University of Manchester

Other organizations : Universidad Complutense de Madrid, University of Leicester, Vanderbilt University Medical Center, University of Liverpool, University of Kent

Top 5 similar protocols

Variable analysis

independent variables

Talin constructs synthesized by PCR using a mouse talin1 cDNA as template
Recombinant His-tagged talin polypeptides
Recombinant His-tagged chicken vinculin domain 1 (Vd1; residues 1–258)

dependent variables

Purified recombinant His-tagged talin polypeptides
Purified recombinant His-tagged chicken vinculin domain 1 (Vd1; residues 1–258)

control variables

Escherichia coli BL21 Star (DE3) used for expression of constructs
LB medium used for culturing Escherichia coli
Nickel-affinity chromatography used for purification of recombinant proteins
AcTEV protease used for removal of His-tag
Anion-exchange chromatography used for further purification

positive controls

Not explicitly mentioned

negative controls

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!