Preparation of the polyclonal antibody against KIAA0649 and its cellular localization

Objective:To prepare and characterize the polyclonal antibody against KIAA0649 and identify the localization and the functional motif of KIAA0649.Methods: Three polypeptides were synthesized based on the bioinformatics analysis of KIAA0649 protein.New Zealand rabbits were immunized with the mixture of the three KIAA0649 peptides coupled with keyhole limpet hemocyanin(KLH).The titer of the antisera was detected with ELISA.The antisera were purified with immuno-affinity chromato-graphy when the titer reached 1∶105.Western blot was performed with the purified antisera on the cell lysates of U2OS cells transfected with either Flag-KIAA0649 or KIAA0649-targeting siRNA.Immunofluorescence was performed with the purified antisera and anti-Flag antibody on the cells transfected with Flag-KIAA0649.A series of Flag-KIAA0649 deletion mutants was constructed by PCR cloning.The cellular compartmentation of full-length Flag-KIAA0649 and its deletion mutants were analyzed with immunofluorescence.Results: The results of Western blot and immunofluorescence demonstrated that the antisera from the KIAA0649 polypeptides-immunized rabbits specifically recognized endogenous and exogenous KIAA0649.The full-length Flag-KIAA0649 displayed specific nuclear foci.The Flag-KIAA0649 deletion mutant containing PENF motif showed the same nuclear foci as the full length of Flag-KIAA0649,suggesting that the PENF motif could be the minimum functional motif of KIAA0649.Conclusion: We have obtained anti-KIAA0649 polyclonal antibody which will be useful for further investigation.The PENF motif could be the minimum functional domain of KIAA0649.