Using 1% SDS in lysis buffer ensures complete solubilization and denatures phosphatases.Contributed by Mike Browning
The use of non-ionic detergents during lysis can fail to fully solubilize cellular proteins and can be the reason an antibody doesn't show a signal 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 samples where synaptic junctions are known to be insoluble in nonionic detergents (Cotman et al. 1974).
1% SDS is needed to completely solubilize proteins.
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. Some researchers use buffers that contain 0.1% SDS such as the RIPA buffer. While this is an improvement over nonionic detergents, it still leaves some proteins in the synaptic junction unsolubilized.
1% SDS also denatures phosphatases, keeping the targets of phosphospecific antibodies phosphorylated.
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 lysis buffer described above has the added benefit that it completely denatures protein kinases and phosphatases.
There are two instances in which 1% SDS is not recommended.
- 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.
- 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.
Want to try our lysis buffer for yourself? You can buy it here: 10X Western Lysis Buffer
While choosing the right lysis buffer is critical to western success, none of this matters if the antibody being used is not reproducible.
For detailed steps on lysate preparation, check out our Lysate Preparation Protocol!
Cotman, W., Banker, G., Churchill, L., and Taylor D. Isolation of post-ynaptic densities from Rat brain (1974) J. Cell Biol., 63 (2) 441-455
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