I have an amyloidogenic peptide and I want study the kinetics of its aggregation by using ThT fluorescence measurement. In order to insure that all the peptides are in monomeric state at the beginning, I would like to know any specific buffer that I can use. But I do not want to use DMSO or HFIP.
It depends on the protein-there is no general method not using DMSO or HFIP (which works for most but not all peptides). Many can be kept stable for a few days at low temperature away from the PI. This works for example for IAPP (pH 5 at 4C), SEVI (low ionic strength any pH), and calcitonin (pH2 below 400 uM) but not amyloid-beta (ph10 at 4C) which still forms oligomers quickly. 1D NMR is useful to distinguish these possibilities. For example, compare:
https://www.researchgate.net/publication/236863949_Alternative_Pathways_of_Human_Islet_Amyloid_Polypeptide_Aggregation_Distinguished_by_F-19_Nuclear_Magnetic_Resonance-Detected_Kinetics_of_Monomer_Consumption?ev=prf_pub
and
https://www.researchgate.net/publication/235748979_Resolution_of_Oligomeric_Species_during_the_Aggregation_of_A1-40_Using_19F_NMR?ev=prf_pub
In the first, the NMR signal corresponding to small species is initially stable than decreases at the same rate as the ThT signal suggesting monomers are rapidly converted to fibers without a stable intermediate species. In the second, the NMR signal drops immediately before fibers are formed indicating the formation of an intermediate species.
Article Alternative Pathways of Human Islet Amyloid Polypeptide Aggr...
Article Resolution of Oligomeric Species during the Aggregation of A...
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