Split-Luciferase Assay for PB1-ANP32A Interaction

Split-luciferase assays were undertaken in 293Ts seeded in 24-well plates. We cotransfected 30 ng each of PB2, PA, and PB1, with the N terminus of Gaussia luciferase (Gluc1) tagged to its C terminus after a GGSGG linker cotransfected using Lipofectamine 3000 along with ANP32A, tagged with the C terminus of Gaussia luciferase (Gluc2) on its C terminus (after a GGSGG linker). Twenty-four hours later, cells were lysed in 100 μl of Renilla lysis buffer (Promega), and Gaussia activity was measured using a Renilla luciferase kit (Promega) on a FLUOstar Omega plate reader. Normalized luminescence ratios (NLRs) were calculated by dividing the values of the tagged PB1 and ANP32 wells by the sum of the control wells, which contained (i) untagged PB1 and free Gluc1, and (ii) untagged ANP32A and free Gluc2 as described elsewhere (15 (link), 37 (link)).

Free full text: Click here

Peacock T.P., Swann O.C., Salvesen H.A., Staller E., Leung P.B., Goldhill D.H., Zhou H., Lillico S.G., Whitelaw C.B., Long J.S, & Barclay W.S. (2020). Swine ANP32A Supports Avian Influenza Virus Polymerase. Journal of Virology, 94(12), e00132-20.

Publication 2020

Renilla lysis buffer Renilla luciferase kit

Anp32a Assays Buffer Cells Lipofectamine Luciferase Luminescence Promega Renilla Renilla luciferase

Corresponding Organization :

Other organizations : Imperial College London, Roslin Institute, University of Edinburgh, Huazhong Agricultural University

Top 5 similar protocols

Variable analysis

independent variables

ANP32A

dependent variables

Gaussia luciferase activity

control variables

Cell line (293T cells)
Transfection method (Lipofectamine 3000)
Lysis buffer (Renilla lysis buffer)
Luminescence measurement (Renilla luciferase kit on FLUOstar Omega plate reader)

controls

Positive control: untagged PB1 and free Gluc1
Positive control: untagged ANP32A and free Gluc2

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!