Acumen Applications - protein kinase activation

Acumen Applications - Protein Kinase Activation
In a whole cell environment, all components required for kinase activation are present in native quantities, thereby eliminating stoichiometric imbalances. The Acumen ^eX3 offers rapid, multicolour scanning and analysis of such assays with normalisation to total cell number when a nuclear stain is applied. It can analyse the entire well area to compensate for variable cell growth or kinase stimulation, with throughputs for 10 minutes for any type of microplate. Validated protocols exist for ERK and JNK which are readily adapted to other kinases or transcription factors. Most use a phosphospecific antibody to signify protein kinase activation and are detected using a FITC or Alexa 488 secondary conjugate.	View showing variable ERK1/2 activation. Green cells are active, red are inactive. (Antibody courtesy of Cell Signalling Technology: www.cellsignal.com )

For cell counting and some translocation assays, a secondary nuclear stain (eg propidium iodide) is added and read in multiplex format. Detection of protein translocation using either GFP-tagged or anti-protein antibodies has been demonstrated for NFkB and SMAD.