Detergent free lysis of small amounts of cells and tissue samples
The first step in purification of proteins from cells or tissue is to disrupt cytoplasmic membrane and release protein and at the same time to limit protein damage by proteolysis and eliminate interfering high molecular mass DNA.Conventional methods of cell lysis employing detergents and/or ultrasonic irradiation could be a challenge when only few milligrams of cells or clinical tissue are available. Our goal was to develop detergent free cell/tissue lysis based on hypotonic shock of small amount of cells compatible with multiple down stream applications (such as ion-exchange and reverse-phase chromatography).
The PCL developed a method for lysis of cultured cells based on hypotonic buffer. The method was applied for express-purification of human elongation factor 2 for assessment of extent of diphthamide modification of His-715. This method will be extended to analysis of cell lines and clinical samples to determine mechanism of resistance to Immunotoxins.