1% SDS is the lysis buffer of choice
for most western blots or
the case of the missing protein in
western blots.
As mentioned in my opening
blog, good antibodies sometimes do not work because of poor technique. One of the most common problems of this type
is the failure to solubilize cellular proteins in the lysis step prior to
western blot analysis. Thus, after centrifugation of the cell lysate many
cellular proteins are discarded with the pellet and are consequently missing (not
detected) from the western blot. This
problem occurs principally because of the use of nonionic detergents such as
NP-40 or triton for cell lysis. These
detergents fail to solubilize many cellular proteins involved in cell
signaling. This problem is particularly
acute in brain where synaptic junctions are known to be insoluble in nonionic
detergents. To obviate this problem, the
lysis buffer of choice for western blots is virtually always 1% SDS which
completely solubilizes membrane and other hard to solubilize proteins and even
synaptic junction proteins. As an added
advantage, SDS also inactivates many cellular proteases. However, inclusion of protease inhibitors
with the 1% SDS is often recommended as some proteases are insensitive to or
even activated (e.g. proteinase K) by SDS.
Phospho-specific antibodies
The use of nonionic
detergents is made even more problematic when the phosphorylation state of a protein
is assayed in western blots using phosphospecific antibodies. This is because
nonionic detergents are ineffective in blocking protein phosphatase
activity. Virtually all cellular lysates
contain high levels of phosphatase activity such that the lysate proteins can
be completely dephosphorylated in a matter of minutes or even seconds. This would make it impossible for a
phosphospecific antibody to work in a western blot as its phosphorylation
target has been removed by the phosphatases.
Fortunately, the 1% SDS lysate buffer described above has the added
benefit that it completely denatures protein kinases and phosphatases.
Exceptions to the rule
1.
Subcellular fractionation and/or protein-protein
interaction.
Because 1% SDS disrupts cell
organelles, it is obviously NOT recommended if isolation of cellular organelles
such as membranes, mitochondria and nuclei is required. However, once the organelles have been
isolated, it is essential that 1% SDS be used to lyse the organelle fraction to
insure solubilization of all the proteins in the organelle. Similarly SDS
solubilization is NOT recommended when analyzing protein-protein interactions
as SDS disrupts these interactions.
2.
Immunoprecipitation.
Antibodies are inactivated
by 1% SDS and this makes immunoprecipitation from the SDS lysis buffer difficult. This effect can be overcome in some cases (Goebel-Goody
et al. 2009) but in the absence of such procedures immunoprecipitation from 1%
SDS is not recommended. Non-ionic
detergents do not typically inactivate antibodies and these detergents are
commonly used prior to immunoprecipitation.
However, it must also be recognized that the lysate prepared by using
nonionic detergent is missing a number of key proteins. So immunoprecipitation from such lysates must
be interpreted with this factor in mind.
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
