Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

The new strategy for co-immunoprecipitation procedure to improve protein-protein interaction study (#954)

Keren Zhang 1 , Yuxing Zhang 1 , Yan Ren 1 , Siqi Liu 1
  1. Clinical Laboratory of BGI Health, BGI, Shenzhen, Beishan Industrial Zone 11th Building, Yantian District, Guangdong, China

During co-immunoprecipitation (Co-IP), the antibodies that can specifically recognize the target proteins, termed as IP-antibodies, are necessary because they have special binding capacity distinct from WB-antibodies that binds to denatured proteins. To seize the transient and weak interactions in a complex, cross-linking with small molecules to some residues in proteins is often adopted, which enables fixation of the interacted structure between target proteins and their bound partners. According to traditional approach, a protein extraction is treated by a cross-linking reagent, then the IP-antibody is added to capture the cross-linked target protein and its interaction proteins. In such experiments, a key issue is how to gain a functional IP-antibody. Because of time- and time-dependence in IP-antibody preparation, Co-IP often encounters a technique barrier lack of proper antibodies. Herein, we raise a question whether WB-antibodies could be used in Co-IP study.

The MS-cleavable linker DSSO was taken to stabilize the interaction complex. The treated protein complexes were denatured by 8M urea to facilitate epitope exposure followed by incubation with WB-antibody. In A549 cell lysate, 22 GAPDH linked proteins were identified with WB-antibody batch, in which most of them are known GAPDH binding proteins. A total of 17 GAPDH linked proteins were found with IP-antibody batch, 5 of them overlapped with the binding components detected by WB-antibody. Surprisingly, number of binding proteins captured by IP-antibody had not reported yet as the interacted proteins with GAPDH. Moreover, one of interacted proteins enriched in WB-antibody batch possessed the transport function, which was consistent with GAPDH functions involved in the vesicle transport.

Thus the preliminary results supported our hypothesis that WB-antibody could be used for identification of protein components upon Co-IP. The strategy might be useful for identifying the binding proteins with low-affinity and at low-abundance.