Lysis buffers: RIPA vs. NETN

The main components of lysis buffers are:

1. a buffer, which creates the environment for the reaction/assay;
2. salt, which creates an ionic environment; and
3. detergent, which acts as a surfactant to break apart membranes.

Different buffers have specific pH ranges which can alter protein confirmation, so it is important to know at which pH your protein of interest is stable. Tris buffer is the buffer component for both RIPA and NETN lysis buffers. Tris buffer has a pH range from 7-9, and RIPA and NETN typically use Tris pH 8. However, if your protein of interest needs a different pH range, a different buffer can be used. NaCl is the salt used in both lysis buffers, but NETN has 250mM NaCl and RIPA has 150mM NaCl. The big difference between RIPA and NETN lysis buffers is the detergents that are used.

There are a variety of different types of detergents that can impact protein extraction and cell lysis. In NETN, NP-40 or Triton X-100 are used, which are non-ionic detergents. These non-ionic detergents will not denature your protein of interest leaving its ability to function intact. RIPA buffer contains NP-40 (non-ionic) and ionic detergents SDS and sodium deoxycholate. This means that RIPA buffer will likely denature the protein target.

In addition to these main components of the lysis buffers, both RIPA and NTEN include EDTA which is a chelating agent. EDTA sequesters cofactors necessary for protease activity reducing potential protein damage and degradation.

RIPA buffer with its ionic detergents is harsher than NETN, which makes it better able to solubilize membrane-bound proteins and nuclear and mitochondrial proteins. However, due to its denaturing properties, RIPA buffer can interfere with downstream applications like immunoprecipitation by disrupting protein folding and protein-protein interactions. NETN buffer is a milder lysis buffer that is frequently used to separate nuclear and cytoplasmic fractions of cells. It is not as effective as RIPA buffer at extracting membrane-bound proteins.

To understand which buffer to pick, you need to know the goal and specifications of your assays. You also need to understand the location and nature of your protein. Will you need harsh cellular treatment or will a milder buffer do? The quality of the downstream applications is based on the efficiency of your protein extraction. The ideal lysis buffer will minimize protein denaturation while maximizing protein solubilization.