Lysis buffer

Buffer creates an environment for isolated proteins. Each buffer choice has a specific pH range, so the buffer should be chosen based on whether the experiment's target protein is stable under a certain pH. Also, for buffers with similar pH ranges, it is important to consider whether the buffer is compatible with the experiment's target protein.[4] The table below contains several most commonly used buffers and their pH ranges.[4]

Lysis buffer usually contains one or more salts. The function of salts in lysis buffer is to establish an ionic strength in the buffer solution. Some of the most commonly used salts are NaCl, KCl, and (NH₄)₂SO_4. They are usually used with a concentration between 50 and 150 mM.[4]

Sodium dodecyl sulfate (SDS) structure

Triton X-100 structure

Detergents are organic amphipathic (with hydrophobic tail and a hydrophilic head) surfactants. They are used to separate membrane proteins from membrane because the hydrophobic part of detergent can surround biological membranes and thus isolate membrane proteins from membranes.[5] Although detergents are widely used and have similar functions, the physical and chemical properties of detergents of interest must be considered in light of the goals of an experiment.

Detergents are often categorized as nonionic, anionic, cationic, or zwitterionic, based on their hydrophilic head group feature.[5]

Nonionic detergents like Triton X-100 and zwitterionic detergents like CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) are nondenaturing (will not disrupt protein functions). Ionic detergents like sodium dodecyl sulfate (SDS) and cationic detergents like ethyl trimethyl ammonium bromide are denaturing (will disrupt protein functions).[6] Detergents are a major ingredient that determines the lysis strength of a given lysis buffer.

One common issue faced by many cell lysis buffers is the disruption of protein structures during the lysis process, partially caused by use of detergents. Detergents often prevent the restoration of native conditions necessary for proper protein folding.[7]

For the longest time, after a detergent-based cell lysis, a buffer exchange and/or dialysis had to be performed to remove the detergent among other hindering compounds to restore native conditions.[8]

To overcome this a solution has emerged in the form of a detergent-free cell lysis buffer. The GentleLys buffer employs copolymers instead of detergents, ensuring efficient cell lysis while maintaining the native environment crucial for the correct folding of cellular components, such as proteins.

Other additives include metal ions, sugar like glucose, glycerol, metal chelators (e.g. EDTA), and reducing agents like dithiothreitol (DTT).[4]

It may be the most widely used lysis buffer. The solubilizing agent is NP-40, which can be replaced by other detergents at different concentrations. Since NP-40 is a nonionic detergent, this lysis buffer has a milder effect than RIPA buffer. It can be used when protein functions are to be retained with minimal disruption.[9]

Recipe:[9]

• 150 mM NaCl
• 1.0% Nonidet P-40 or Triton X-100
• 50 mM Tris-Cl
• Adjust pH to 7.4

RIPA buffer is a commonly used lysis buffer for immunoprecipitation and general protein extraction from cells and tissues. The buffer can be stored without vanadate at 4 °C for up to 1 year.[10] RIPA buffer releases proteins from cells as well as disrupts most weak interactions between proteins.[9]

Recipe:[10]

• 1% (w/w) Nonidet P-40 (NP-40)
• 1% (w/v) sodium deoxycholate
• 0.1% (w/v) SDS
• 0.15 M NaCl
• 0.01 M sodium phosphate, pH 7.2
• 2 mM EDTA
• 50 mM sodium fluoride (NaF)
• 0.2 mM fresh sodium orthovanadate (Na₃VO₄.2H₂O, it has phosphatase inhibitor function because it mimics phosphate)
• 100 U/ml protease inhibitor, such as aprotinin

SDS is ionic denaturing detergent. Hot SDS buffer is often used when the proteins need to be completely solubilized and denatured.

Recipe:[10]

• 0.5% (w/v) SDS
• 0.05 M Tris⋅Cl
• Adjust pH to 8.0
• Add 1 mM fresh dithiothreitol (DTT)

ACK is used for lysis of red blood cells in biological samples where other cells such as white blood cells are of greater interest.[11]

Recipe:[12][13]

• 150 mM ammonium chloride
• 10 mM potassium bicarbonate
• 0.1 mM EDTA
• Adjust pH to 7.2-7.4

The GentleLys buffer employs synthetic nanodisc copolymers to gently disrupt the cell membrane, offering a milder alternative to conventional detergent-based lysis buffers. This gentle approach eliminates the need for harsh chemicals, creating an environment that preserves the native state of cellular proteins. Consequently, the proteins maintain their structural integrity and functionality, a marked departure from the denaturing effects of detergent-based buffers.