But which detergent do you put in your lysis buffer? Think carefully because the detergent you choose could mean the difference between a blank blot and an image worthy of the highest tier journals.
Here are a few hints to help you choose the right lysis buffer:
Rip it open with RIPA
RIPA buffer is what you want to use if you want to solubilize all membranes. Lyse your cells with this buffer and you will release all proteins within compartments, including nuclear and mitochondrial proteins. This is due to the combination of harsh denaturing, ionic detergents (sodium deoxycholate and SDS) and the milder, nonionic detergent (NP-40).
Watch out! If you want to study protein-protein interactions, then RIPA buffer is not for you! RIPA will destroy any protein interactions and cannot be used for co-immunoprecipitations (co-IPs).
Also be forewarned – when you solubilize nuclear membranes with RIPA buffer, genomic DNA is released. It will turn your sample into a gooey mess and be very difficult to pipette. You can shear the DNA by passing the sample through a needle or by spinning it through a homogenizer spin column (my personal trick).
RIPA lysis buffer: 25mM Tris•HCl, pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS)
Maintain some integrity with NP-40 or Triton X-100 lysis buffer
NP-40 (Nonidet P-40) and Triton X-100 are milder, nonionic detergents. They are good at solubilizing membrane proteins and for isolating cytoplasmic proteins. Proteins retain their native state in the presence of these detergents and protein-protein interactions can be preserved. These buffers can be used for co-IPs.
NP-40 and Triton X-100 will not lyse nuclear membranes. After lysis, pellet the nuclei by centrifugation and transfer the supernatant to a new tube. If you wish to isolate both the nuclear and soluble fractions, resuspend the nuclear pellet in RIPA buffer.
NP-40 is also marketed under the name Igepal CA-630.
NP-40/Triton X-100 lysis buffer: 50 mM Tris•HCl, pH 8.5, 150 mM NaCl*, 1% detergent*
*The concentrations of salts and detergents can be altered to optimize protein recovery.
And the other detergents?
Every protein is different and interacts with detergents in different ways. If you don’t think you are solubilizing all of your protein or if you are looking for a specific protein-protein interaction, then you may want to play around with different detergents.
CHAPS (3- [(3-cholamidopropyl)dimethylammonio] -1-propanesulfonate; a zwitterionic detergent) is harsher than the nonionic detergents, but milder than the ionic detergents. Therefore it can maintain some protein-protein interactions, but may be better at solublization.
Digitonin is a mild, noninonic detergent similar to NP-40 and Triton X-100. It is very effective at solubilizing membrane proteins. If you are looking for a protein-protein interaction using co-IPs, then you might want to try digitonin. When I was a graduate student I worked with two proteins that would only co-IP in the presence of digitonin.
To use these detergents begin at a 1% concentration.
Putting it to work for you
All this sounds good, but practically speaking, what should you do? For most every day Western experiments, I keep bottles of RIPA and NP-40 lysis buffers in the refrigerator. If I am working with a nuclear protein, I use RIPA buffer, otherwise I try NP-40 buffer. If I am not having good results with NP-40, then I move on to RIPA.
If I am doing co-IPs, I will usually compare results after lysing in 2 different detergents: NP-40 and either digitonin or CHAPS.
Whatever buffer you choose, don’t forget to add the protease inhibitors!
Photo courtesy of Matt.