High sensitivity and in many cases sufficient intrinsic label concentrations of

High sensitivity and in many cases sufficient intrinsic label concentrations of either naturally occuring tryptophanes or genetically engineered fluorescent tags [6,7,41,42]. 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 tert-Butylhydroquinone 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 samplesFast Proteolysis Assay FASTppwithout 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 [6,43?5]. 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 [1]. Several features of FASTpp differ significantly from Pulse Proteolysis: 1. The rapid temperature increase in FASTpp significantly increases the denaturation rate of kinetically-stable proteins compared to urea titrations at room temperature, e.g. for ligand-bound maltose binding protein [1]. 2. High temperature (up to 80uC) has little effect on the intrinsic proteolysis rate; high urea concentrations however inhibit the enzyme [1]. 3. Temperature gradients reveal quickly self-aggregating unfolded species while urea may dissolve aggregates. Taken together, both approaches have complementary benefits: FASTpp gives insight into thermal stability, Pulse Proteolysis into equilibrium unfolding. FASTpp, however, requires less experimental time. Considering the broad range of folds that can be analysed by FASTpp and the specificity, robustness and speed of the method, we anticipate a broad range of future applications. Minimal sample preparation requirements and use of standard K162 site molecular biological techniques allow applications in protein engineering, cell biology and biomedical research.coommassie upon binding to protein was measured and the integrated fluorescence intensity per protein band was compared to the corresponding two-fold dilution series of undigested proteins of known concentration to fit the parameters of a second-order polynom describing the dependence of fluorescence on protein concentration [23].Determination of the temperature dependence of the intrinsic proteolysis rate of TLWe determined temperature dependence of TL activity analogous to a previous approach for monitoring urea dependence of TL activity [1]. 1326631 Briefly, we used 6 nM and 3 nM TL to cleave a fluorigenic model substrate (ABZ-Ala-Gly-Leu-Ala-NBA) to monitor the reaction by fluorescence dequenching of this substrate at various temperatures. For quantification we used a pseudo-firstorder kinetic model that assumes a constant concentration of the catalyst (TL) over the course of the experiment and full accessibility of the substrate. As fluorescence increases relative to the extent of dequench, we fitted the intrinsic rate by using the formula: F F0 max {F0 ?1{e kt?F is fluorescence, F0 is the initial fluorescence, Fmax is the fluoresc.High sensitivity and in many cases sufficient intrinsic label concentrations of either naturally occuring tryptophanes or genetically engineered fluorescent tags [6,7,41,42]. 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 samplesFast Proteolysis Assay FASTppwithout 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 [6,43?5]. 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 [1]. Several features of FASTpp differ significantly from Pulse Proteolysis: 1. The rapid temperature increase in FASTpp significantly increases the denaturation rate of kinetically-stable proteins compared to urea titrations at room temperature, e.g. for ligand-bound maltose binding protein [1]. 2. High temperature (up to 80uC) has little effect on the intrinsic proteolysis rate; high urea concentrations however inhibit the enzyme [1]. 3. Temperature gradients reveal quickly self-aggregating unfolded species while urea may dissolve aggregates. Taken together, both approaches have complementary benefits: FASTpp gives insight into thermal stability, Pulse Proteolysis into equilibrium unfolding. FASTpp, however, requires less experimental time. Considering the broad range of folds that can be analysed by FASTpp and the specificity, robustness and speed of the method, we anticipate a broad range of future applications. Minimal sample preparation requirements and use of standard molecular biological techniques allow applications in protein engineering, cell biology and biomedical research.coommassie upon binding to protein was measured and the integrated fluorescence intensity per protein band was compared to the corresponding two-fold dilution series of undigested proteins of known concentration to fit the parameters of a second-order polynom describing the dependence of fluorescence on protein concentration [23].Determination of the temperature dependence of the intrinsic proteolysis rate of TLWe determined temperature dependence of TL activity analogous to a previous approach for monitoring urea dependence of TL activity [1]. 1326631 Briefly, we used 6 nM and 3 nM TL to cleave a fluorigenic model substrate (ABZ-Ala-Gly-Leu-Ala-NBA) to monitor the reaction by fluorescence dequenching of this substrate at various temperatures. For quantification we used a pseudo-firstorder kinetic model that assumes a constant concentration of the catalyst (TL) over the course of the experiment and full accessibility of the substrate. As fluorescence increases relative to the extent of dequench, we fitted the intrinsic rate by using the formula: F F0 max {F0 ?1{e kt?F is fluorescence, F0 is the initial fluorescence, Fmax is the fluoresc.

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