Identification with 2D-western blotting, extraction and antibody preparation of HSP70

ObjectiveTo validate the expression level of HSP 70 in the tissues of lung cancer,gain the human HSP 70 from lung cancer tissues and prepare its antibody,lay the foundation for further research aboutits biological function and clinical use.MethodsLung cancer tissues and cancerous lung cancer were collected,their expression level of HSP 70 were validated by using two-dimensional western blotting and it was separated by ConA-sepharose affinity column chromatography and EDTA negative charge column chromatography.HSP 70 extracted from lung cancer was used as antigen to biopan human scFv phage library of lung cancer through four rounds of biopanning.High affinity and specificity positive monoclonical phage antibody was identified by ELISA.ResultsTwo-dimensional western blotting results showed the expression level of HSP 70 of lung cancer tissue was separated and purified.HSP 70 was used as antigen to biopan human antibody library,then its antibody was prepared.ConclusionThe gaining of human HSP 70 and its human antibody lay the foundation for further research aboutits biological function and clinical use.