Conversely, proteins containing large internal disordered regions are expected to be cleaved by default – unless they fold for GDC-0449 instance by a coupled folding and binding mechanism in vivo. Accurate disorder predictions for watersoluble proteins such as PONDR-Fit might therefore be useful to preselect suitable candidate proteins for FASTpp assays and guide the data interpretation. We established FASTpp as a biophysical tool to monitor structural protein stability for both isolated proteins and in lysate. We observed high intrinsic protease activity over a large temperature range from physiological temperatures to 80uC in agreement with previous related studies. An even more thermostable TL variant may extend FASTpp to extremely thermostable substrates. We investigated possible applications of FASTpp for interactions of a folded protein with ligand in either presence or absence of cellular lysate. We obtained an about 10uC higher temperature of unfolding for the ligand saturated MBP in both cases. This agrees qualitatively with previous DSC studies, where MBP unfolded at 55uC and 65uC in maltose-bound form at a heating rate of 1uC/min. It also agrees qualitatively with our data obtained by intrinsic protein fluorescence. The differences of absolute values are likely due to different timescales of heating and the fact that unfolded protein is removed from the equilibrium in the FASTpp assay. Presence of lysate had a stabilising effect on apo MBP as monitored by FASTpp while in case of RNAse H stability analysis by Pulse Proteolysis, diluted lysate did not affect the protein stability, possibly due to dilution by urea. Can we determine absolute thermal melting points of proteins by FASTpp? The determination of absolute Tm values requires equilibrium conditions, which can be achieved in particular by calorimetric methods. In FASTpp, the unfolding temperature values depend on the experimental conditions such as temperature range, heating rates, protein concentration and protease susceptibility of the protein of interest. This allows the precise relative stability analysis of point mutations, ligand binding and different environments including cell lysates. What method should be chosen for which application? Fluorescence is widely used due to its high sensitivity and in many cases sufficient intrinsic label concentrations of either naturally occuring tryptophanes or genetically engineered fluorescent tags. FASTpp is a useful complementation to fluorescence-based assays in cases where intrinsic labels are below detection levels or genetic manipulation is not possible. The specific advantage of FASTpp, however, is its ability to analyse protein stability at low concentrations and in complex solutions, such as lysates and primary patient samples. Specific antibodies allow stability analysis by FASTpp of cell or tissue-derived samples without the need for tagging or purification. To investigate possible links between biophysical and pathological mechanisms of tumour mutations, patient tissues may be analysed for putative stability changes in disease-related proteins such as kinases and tumour suppressors. FASTpp experiments can be done in laboratories equipped with standard biochemistry instruments and do not require advanced biophysical equipment. FASTpp is also an alternative for Pulse Proteolysis. In this ex vivo assay, equilibrium unfolding at room temperature in urea precedes a short proteolysis pulse to probe unfolding. Several features of FASTpp differ significantly from Pulse Proteolysis: 1.