Determining Fusion Protein Antiserum Quality by Staining of Transfected Drosophila S2 Cultured Cells

INTRODUCTION

Once antibodies against a Drosophila protein have been produced and an antiserum is determined to have a high titer against the fusion protein used for immunization, the next step is to ask whether the antiserum recognizes the appropriate antigen in Drosophila tissues. This protocol describes staining of transfected S2 cells for testing antisera. There are several advantages to using transfected S2 cells as a first tissue test. First, the antibody only needs to penetrate a single cell. In addition, these cells are easy to fix and stain and require no dissection. Second, the overexpressed protein will be easier to detect than lower levels of endogenous protein. Third, background is generally less of a problem in S2 cells than in embryonic or imaginal tissues, allowing lower dilutions of the antiserum to be tested if the response is marginal. Finally, the untransfected cells form a useful built-in control. S2 cell stainings are also an easy way to determine the subcellular localization of a particular protein.