The extraction of membrane proteins is a special case of protein extraction. Detergents are often used in the extraction and purification of membrane proteins, which dissolve membrane proteins with the help of detergents.

Detergents are amphoteric bipolar small molecules that are hydrophilic at one end and hydrophobic at the other, and can be dissolved in water. In addition to cholates, detergent molecules are usually composed of linear or branched hydrocarbon tails and hydrophilic heads with varying structures.

An important property of detergents is the ability to form a molecular clump structure, with clusters of detergent molecules pointing the hydrophilic heads outward. It generally exists in the form of colloidal particles in aqueous solutions. The dissolved membrane protein binds to the detergent molecule to form a clump, and the hydrophobic or transmembrane region of the membrane protein is covered by the detergent molecule, making it soluble with aqueous buffer.

The lowest concentration of micelles that can form micelles is called critical micelle concentration (CMC), and after the CMC value is higher, the detergent will aggregate to varying degrees, and even aggregate into a phase alone when the concentration reaches a certain level. Detergents with high CMC values form monomer molecules when diluted, which can be quickly removed by dialysis. In addition, the molecular weight of the microgels formed by the detergent is very important for dialysis, gel filtration chromatography, and native electrophoresis. Temperature, pH, ionic strength, the presence of polyvalent metal ions, and the purity of organic solvents and detergents can all affect the CMC value of detergents.

Ideally, the choice of detergent should not only consider its purity, as well as the cost when applied in large quantities, but also whether the amount of detergent should be sufficient to adequately dissolve the protein without irreversible denaturation.

In addition, how to separate excess detergents from dissolved membrane protein fragments is another criterion for detergent selection. The use of detergents to extract membrane proteins from biofilms should comprehensively consider the effectiveness, purity, cost and source of detergents.

Although high concentrations of detergents tend to cause protein denaturation, proteins can be restored after removal. To avoid protein denaturation, a nonionic detergent with a concentration of less than 0.1% can be used. The characteristics of commonly used detergents are shown in the table below:

(1) Ionic descaler

Ionic detergents include a charged head, the cationic charge is the cationic detergent, and the anionic charge is the anionic detergent. The disadvantage of this type of detergent is that it makes the protein highly denaturated, but the use of this type of detergent allows the protein to be separated in the form of monomers, which is more convenient for determining the molecular weight of the protein.

(1) SDS or lithium lauryl sulfate (LiDS)

These are two anionic detergents, and LiDS has the advantage of being soluble at 4°C, while SDS is insoluble. To completely dissolve 1 mg of membrane protein, 10 mg or more of LiDS is required.

It is important to note that the use of this detergent in buffers containing potassium ions and buffers containing ammonium sulfate is prone to precipitation at room temperature. The concentration of salt ions in solution can significantly affect the CMC value of the detergent, such as SDS, when the concentration of NaCl is 0~0.5mol/L, its CMC value decreases from 8mmol/L to 0.5mmol/L.

(2) Sodium cholate and sodium deoxycholate

Sodium cholate and sodium deoxycholate are anionic detergents, which have a weak effect on protein denaturation compared with other ionic detergents.

Detergent concentrations higher than critical micelle concentrations can form two types of molecular groups: primary micelles (including 9 molecules) and secondary micelles (including 9~60 molecules). Unlike other detergents, free cholate and deoxycholate monomer molecules above the critical micelle concentration value can accumulate. The pKa of this type of detergent molecule is 8~9, and when the pH is below 7.5, the acidic form of the detergent molecule will precipitate. Divalent cations can also precipitate sodium deoxycholate.

(2) Non-ionic detergents

Nonionic detergents have non-polar hydrophilic heads, which have weak interference with protein-protein interactions, which is more beneficial for separating functional protein complexes. Compared with ionic detergents, their denaturation effect on proteins is weaker, but proteins are prone to aggregation in this detergent.

(1) Qulaton X-100

When the mass concentration of Tralaton X-100 is 1%~3% (mass fraction), the activity of many proteins remains unchanged. For membrane proteins, 10 times or higher concentrations of Tralaton X-100 are used to dissolve. Trathon X-100 has a strong UV absorption value at 280nm.

(2) Qulatong X-114

When the temperature is above 20°C, the addition of 2% (mass fraction) of Tratone X-114 to the protein solution allows the protein to be separated, with the soluble protein still in the liquid phase and the membrane protein entering the detergent phase.

(3) Capryglucoside

Compared to tralatone X-100, capryglucoside has many advantages. 20~45mmol/L capryglucoside is enough to dissolve membrane proteins; Capryglucoside has a high CMC value, so it is easily removed from solution.

(4) Tween 20

Tween 20 is commonly used in solid-phase immunocytochemical reactions such as western blotting to block non-specific protein interactions. When doing Western blotting, blocking the unbound site on the membrane with Tween 20 can reduce the non-specific binding of resistance. Compared to Tween 80, Twain 20 is milder.

(3) Amphoteric detergent

Compared with non-ionic detergents, amphoteric detergents have positive and negative ion heads, which can reduce the interference of protein and protein interaction, and the effect of ionic detergents on protein denaturation.

Commonly used zwitterionic detergents include CHAPS{3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid} and Zwittergent3-14, among which CHAPS does not interfere with ion exchange chromatography and isopointal focused electrophoresis, and CHAPS-containing protein solutions can be cryopreserved.