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. 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. 

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 lysis 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.

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!

Using 1% SDS and still ending up with goop? Check out our blog on The Truth about Goopy Lysate!

More Protocols: We also have in-depth protocols for Western Blotting and Phosphatase Treatments.

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
Summary
1% SDS is the lysis buffer of choice for most western blots
Article Name
1% SDS is the lysis buffer of choice for most western blots
Description
Using 1% SDS (an ionic detergent) in the lysis step prior to Western blotting completely solubilizes membranes and other hard to solubilize cellular proteins.
Author
Mike Browning
Publisher Name
PhosphoSolutions
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10 thoughts on “1% SDS is the Lysis Buffer of Choice for Most Western Blots

  1. When you use non-ionic detergents for lysis, you can include 150 mM NaCl to help retain proteins that would normally be lost without salt with non-ionic detergents. This will give you a better isolation for Co-IP experiments. The buffer I use is: 1% Triton X-100, 50 mM Tris pH 8.0, 150 mM NaCl + 1x protease inhibitors + 5 mM EDTA.

    An added benefit of using this buffer is that it doesn’t lyse the nucleus, thus preventing the lysate from becoming overly viscous from all of the DNA.

    1. You are quite right Darius that non-ionic detergents require NaCL to work effectively. However, remember this buffer will not solublize the synapse and thus in CO-IP the pellet will artifactually contain synaptic proteins. This can be avoided by preclearing the lysate by centrifugation before adding the antibody. Of course then synaptic proteins cannot be accurately studied in CO-IP of such a lysate. Obviously this is primarily a brain problem.

      1. Thanks for your question even if it is a difficult one to answer. One difficulty I face is that I have little experience with nuclei. So DO NOT assume this answer is coming from an expert. Having said that my reading of the literature suggests a number of issues to keep in mind. Many of these issues are described in a classical paper on sub-cellular fractionation and protein extraction (Nature Protocols, 1(4), 1872, 2006). This paper describes initial enrichment of the nuclear and other fractions. Such a method should help to increase the signal to noise in your Co-IP but be aware that redistribution of proteins can occur in this procedure. Second the paper describes various protein extraction procedures and argues that DNAase extraction may be optimal for co-IP experiments. There are also many companies that offer nuclear extraction cocktails. As I mentioned we have no experience in this area and so we cannot evaluate these procedures. However, as with any “proprietary cocktail” be sure to “do your bookkeeping”. By that I mean you should assay all your fractions along the way to keep track of your target proteins. This way you will know whether a particular treatment give a good recovery of your target (and potential CO-IP target). Good luck on this. I am sorry if I am telling you stuff you already know but I couldn’t determine your expertise level from your question. If you would like additional info on preparing the tool you use to pull down the antibody I have a bit of experience in using SAC cells (I know this is dated) that also likely pertains to more recent pull down tools.

  2. Dear Mr Browning,

    we also have used your recommended 1% SDS lysis buffer successfully. However, we have some problems with bubbles while mixing with the loading buffer (Blue Loading Buffer, Cell Signaling) but also during protein quantification, thus we must always work very carefully. Have you ever tested lower SDS concentrations with your lysis buffer or do you have some advice for the handling the 1% SDS Lysis buffer?

    Sincerely
    L. Konrad

    1. Lower percentages of SDS in your lysis buffer will not eliminate the bubbles, and you run the risk of incomplete solubilization of synaptic junction and membrane proteins. Also, since your loading buffer has 6% SDS, a lower amount of SDS in your lysis buffer will not have much impact on the bubbling issue. If you are experiencing bubbling when mixing with the loading buffer, consider rocking and pipetting slowly to avoid air bubbles. If using a vortexer, do it on slower speed. Be sure to spin down any bubbles before loading your gel. When we quantify proteins, we use Pierce’s BCA Protein Assay, and due to the high concentrations of lysate, we typically only need 1-5 uL of lysate in 2 mL of Assay Buffer.
      If you are seeing bubbling during homogenization or sonication of your original lysate in simple lysis buffer, the following steps should help. With homogenization make sure the size of the homogenizer is small enough so that the pestle when homogenizing never rises above the level of the liquid. In addition attach the pestle to a drill and use it turn the pestle slowly but steadily without any up and down motion. With a sonicator, make sure the sonicator tip is chilled prior to use, and once you begin sonicating, make sure the tip stays completely submerged. You may need to use very small probes and also small microfuge tubes to effect this. Also try reducing the frequency and amplitude of bursts on your sonicator.

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