Western Blotting technique standardization of protein quantification after PIPKIIa enzyme gene overexpression in human erythroid cell cultures

• Grant number: 15/04179-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2015
• End date: December 31, 2015
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Maria de Fatima Sonati
• Grantee: Thais Traina Jorge
• Host Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 14/00984-3 - Red blood cell disorders: pathophysiology and new therapeutic approaches, AP.TEM

Abstract

The Western blotting (WB) is an important tool used in cell and molecular biology for studying proteins and it is considered highly sensitive and specific. Once proteins are bound to membranes, they are accessible for immunological or biochemical assays. The WB protocol involves selection of the appropriate method, apparatus, membranes, buffers and transfer conditions. The aim of this project is to standardize the WB technique for quantifying of PIPKIIA protein and alpha, beta and gama globins after overexpression, by RNA-induced gene activation technology (RNAa), of PIPKIIA gene in cultures of human erythroid cells. Thus, CD34+ cells transduced by lentiviral vectors will be lysed and their proteins extracted and measured by the WB technique to be standardized. The protein analyzes, performed using this technique, will be designed to estimate the relative amount of globin chains and PIPKII±, after the procedure of gene overexpression, correlating the results with those obtained in the qPCR. We expect to observe a possible relationship between PIPKIIA and globin production in erythroid cells in an attempt to elucidate the functions of this enzyme and evaluate its role in the (direct or indirect) regulation of globin genes.