My 26 aminoacid peptide, known to immediately trigger pore formation on the plasma membrane of the cells leading to more than 90% cell death (confirmed by both MTS and LDH assay) following its incubation at 1-10ug/ml concentration for 24 hours, showed reduced cell death (30 -40%) at concentrations above 50 ug/ml. What could be the possible reason for this non-linearity? How to confirm/study what is going on in this case? The peptide are known to form tetramers following attachment to the bilayers of the plasma membrane, right before forming toroidal pores in the membrane resulting in the leakage of the cell constituents.
In high concentratiions they do not pass thruogh the cell membrane
First i'd like to say that i know very little about this subject. But i could imagine a mechanism whereby the low concentration kills of cells over the 24h period.( you check for cell death at the end of the 24h period right ?) Whereas a higher concentration perhaps kills off the cells quicker, allowing the remainder of the cells to proliferate hence giving the impression of reduced cell death when you measure cell death after 24h. Not sure if this makes any sense, but i figured i'd put it out there.
Best,
Pieter
could your peptide be forming micelles at higher concentrations? If that is the case it may not be attaching to the membrane at the higher concetrations.
Could your peptide form micelles at higher concentrations? if it does it could inhibit attachment to the plasma membrane.
It may happen something similar to doxycyclin regarding its citotoxicity. It decreases after 1 week of incubation, without modifying the concentration: the reason is the formation of doxycycline aggregates with lesser activity. Your polypeptide may form octamers or sexadecamers or any other kind of multitetramers that loose activity on the membrane. Have you run any bioinformatic simulation on the tridimensional structure of the 26 amino acid peptide at low and high concentrations?
I guess that you have a problem of peptide solubility and/or formation of oligomers with limited access to membranes
you may have additives problems.
your peptides could undergo a drop in solubility
Your peptide can form micelles.
Please assume thta you will use others assay of cell proliferation than MTT (MTS)... If you can do some flow cytometry, this will be more adequat
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