Preparation Of Gas8 Polyclonal Antibody And Screening Of Interacting Proteins With Gas8

Objective: Gas8 (growth arrest-specific8) protein is expressing one of the growth arrest-specific genes. The aim of this study is to prepare Gas8 polyclonal antibody and check the expression of Gas8 protein in the tissues of the mouse, and select the Gas8 interacting proteins through the yeast two-hybrid system. Methods: His-tagged Gas8 was expressed in E.coliBL21(DE3). Rabbit Gas8 polyclonal antiserum was generated by using the purified recombinant His-tagged Gas8 inclusion body as the antigen, and further separated by affinity purification. To confirm the specificity of the Gas8 antibody, transiently expressed Flag-Gas8 fusion protein was analyzed by Western blot with anti-Flag monoclonal antibody as control. The produced antibody was used to check the expression of Gas8 protein in mouse tissues by Western blot. We use Gas8 as bait protein to screen the related protein by yeast Two-hybrid System. Results: The results showed that the specific anti-Gas8 antibody was obtained and it could be used to detecte the expression of Gas8 protein in mouse tissues. No positive clone was identified by cdc25H yeast Two-hybrid System. Three positive clones were identified by AH109 yeast Two-hybrid System.Conclusions: Antibodies specifically binding to Gas8 could be obtained by immunization, and expression of Gas8 was detected in heart, lung, kidney, brain, liver, spleen, muscle and testis of mouse with the generated antibody. The interaction of RARRES2 , CUTC and EIF2B1 protein with Gas8 were tested by yeast mating.