Preserving Phosphorylation States in Lysates

When using antibodies to study specific proteins and especially phosphoproteins in western blots of cell lysates the most suitable lysate buffer is virtually always just 1% SDS. This is because non-ionic detergents (e.g. triton, np-40, CHAPs) fail to fully solubilize all proteins and in brain they fail to solubilize the synaptic junction proteins.

Moreover, since these non-ionic detergents do not denature most proteins, extremely rapid (msecs) alterations in phosphorylation will occur in nonionic detergent lysates. This can be somewhat minimized with phosphatase and kinase inhibitors but these agents are never as effective as SDS. Given that SDS-PAGE is the subsequent use for the lysate SDS solubilization is thus the method of choice for preparing lysates for western blots.

Refs:

Davies, KD, Goebel-Goody, SM, Coultrap, SJ and Browning, MD (2008) Long-term synaptic depression that is associated with GluR1 dephosphorylation but not AMPA receptor internalization. J Biol Chem.283:33138-46. Goebel-Goody, SM, Davies, KD, Linger, RA, Freund,R and Browning, MD. (2009) Phospho-regulation of synaptic and extrasynaptic NMDA receptors in adult hippocampal slices. Neuroscience 158:1446-1459