Purification and Identification of Influenza NP Interactors

The nucleoprotein of A/HK/156/97 (H5N1) was expressed as a maltose binding protein (MBP) fusion protein as described previously [8 (link)]. After RNase A and thrombin treatment, purified NP was coupled to NHS beads. The crude lysate of H292 cells was allowed to flow through immobilized NP. After extensive washing, bound proteins were eluted with 2 M NaCl, separated through 2D electrophoresis, and visualized via silver staining. Surplus spots compared with the control (activated–deactivated NHS beads without immobilized NP) were excised for MALDI–TOF MS/MS analysis. A combined (MS + MS/MS) analysis was carried out against the NCBI non-redundant database using the ProteinScape 3.0 software from Bruker, Billerica, MA, USA.

Free full text: Click here

Tang Y.S., So W.K., Ng K.L., Mok K.P, & Shaw P.C. (2022). Interaction of Influenza A Nucleoprotein with Host hnRNP-C Is Implicated in Viral Replication. International Journal of Molecular Sciences, 23(21), 13613.

Publication 2022

ProteinScape 3.0

Electrophoresis H5n1 Hk 156 Maldi Maltose binding protein Ms ms Nacl Nucleoprotein Proteins Rnase Spots Thrombin

Corresponding Organization : Chinese University of Hong Kong

Other organizations : Hong Kong Jockey Club

Top 5 similar protocols

Variable analysis

independent variables

Expression of nucleoprotein (NP) of A/HK/156/97 (H5N1) as a maltose binding protein (MBP) fusion protein
Coupling of purified NP to NHS beads
Allowing the crude lysate of H292 cells to flow through immobilized NP
Elution of bound proteins with 2 M NaCl

dependent variables

Proteins bound to immobilized NP
Proteins separated through 2D electrophoresis
Proteins visualized via silver staining
Proteins identified through MALDI-TOF MS/MS analysis

control variables

RNase A and thrombin treatment of purified NP
Activated-deactivated NHS beads without immobilized NP

controls

Activated-deactivated NHS beads without immobilized NP as a negative control

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!