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Lysis buffer

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Draft edition for Lysis buffer:

Lysis Buffer Overview:

A lysis buffer is a buffer solution used for the purpose of lysing cells for use in molecular biology experiments that analyze the compounds of the cells (e.g. western blot). Most lysis buffers contain salts (e.g. Tris-HCl or EDTA) to regulate the acidity and osmolarity of the lysate. Sometimes detergents (such as Triton X-100 or SDS) are added to break up membrane structures. Lysis buffer can be used on both animal and plant tissue cells.

In studies like DNA fingerprinting the lysis buffer is used for DNA isolation. Dish soap can be used in a pinch to break down the cell and nuclear membranes, allowing the DNA to be released. Other such lysis buffers include the proprietary Qiagen product Buffer P2.

Choose the Optimal Buffer:

Lysis buffer usually contains a combination of salts. The common salts used are urea, tris-HCl, NaCl, EDTA, etc.. It is essential to choose the best buffer based on the purpose and design of the experiments. The important factors to be considered are: pH, ionic strength, usage of detergent, preventative measure for proteolytic processes. For example, detergent addition is necessary when lysing Gram-negative bacteria, but not for Gram-positive bacteria. It is common that protease inhibitor is added to lysis buffer, along with other proteolytic inhibitors of choice, such as phosphotase inhibitor.

Detergent:

Detergents are organic amphipathic surgactants. They are used to dissolve membrane proteins because biological membranes can form micelles with the hydrophobic part of detergent and thus isolate membrane proteins from membranes. Although detergents are widely used and have similar functions, it is important to understand the physical and chemical properties of the detergents of interest in order to determine the optimal one to use for experiment.

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

Detergents can also be categorized as nondenaturing and denaturing. The former contains nonionic detergents like Triton 100X and zwitterionic detergents like CHAPS; the latter contains ionic detergents like sodium dodecyl sulfate (SDS) and cationic detergents like ethyl trimethyl ammonium bromide.

Commonly used lysis buffers:

RIPA (RadioImmunoPrecipitation Assay) lysis buffer

RIPA buffer is another commonly used lysis buffer for immunoprecipitation and general protein extraction from cells and tissues. As the name suggests, the buffer is names after its usage.

Recipe:

• 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
• 0.2 mM sodium vanadate added fresh from 0.2 M stock solution
• 100 U/ml aprotinin (Trasylol, Pentex/Miles)
• Store buffer without vanadate at 4°C up to 1 year

SDS (sodium dodecyl sulfate)

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

Recipe:

• 0.5% (w/v) SDS
• 0.05 M Tris⋅Cl, pH 8.0
• 1 mM DTT, added fresh

O’Farrell’s lysis buffer

This buffer is good in dissolving more hydrophilic proteins, but not as good in dissolving highly hydrophobic proteins including many of the membrane proteins.

Recipe:

• 9.5M urea

• 2–4% CHAPS
• 1% dithiothreitol
• 2% [v/v] carrier ampholytes

Mild lysis buffer

Recipe:

• 10 mM 3[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) or 1% (w/w) Nonidet P-40 (NP-40)
• 0.15 M NaCl
• 0.01 M sodium phosphate, pH 7.2 (appendix 2)
• 2 mM EDTA
• 50 mM sodium fluoride
• 0.2 mM sodium vanadate added fresh from 0.2 M stock solution
• 100 U/ml aprotinin (Trasylol, Pentex/Miles)
• Store buffer without vanadate at 4°C up to 1 year

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