Sonics VCX750 Bedienungsanleitung PDF herunterladen (Seite 27)

Detergent cell lysis is sometimes used in conjunction with ultrasonic processing to

facilitate disruption. Detergents break the lipid barrier surrounding cells by solubilizing

proteins and disrupting lipid:lipid, protein:protein and protein:lipid interactions.

Detergents, like lipids, self-associate and bind to hydrophobic surfaces. They are

composed of a polar hydrophilic head group and a nonpolar hydrophobic tail and are

categorized by the nature of the head group as either ionic (cationic or anionic),

nonionic or zwitterionic. Their behavior depends on the properties of the head group

and tail.

NOTE:

When working with detergents, do not use a probe with a replaceable tip. Use a solid

probe instead.

Unfortunately, there is no standard protocol available for selecting a detergent to use

for membrane lysis, the ideal detergent will depend on the intended application. In

general, nonionic and zwitterionic detergents are milder and less denaturing than ionic

detergents and are used to solubilize membrane proteins when it is critical to maintain

protein function and/or retain native protein:protein interactions for enzyme assays or

immunoassays. Zwitterionic detergents and nonionic detergents are commonly used

for these purposes. In contrast, ionic detergents are strong solubilizing agents and

tend to denature proteins, thereby destroying protein activity and function. There are

a few commonly used ionic detergents that are only mildly denaturing, including

sodium cholate and sodium deoxycholate.

The choice of detergent for cell lysis also depends on sample type. Animal cells,

bacteria and yeast all have differing requirements for optimal lysis due to the presence

of absence of a cell wall. Because of the dense and complex nature of animal tissues,

they require both detergent and sonication. In addition to the choice of detergent,

other important considerations for optimal cell lysis include the buffer, pH, salt

concentration and temperature. Consideration should be given to the compatibility of

the chosen detergent with downstream applications. For example, if the detergent

used for lysis must be removed, then a dialyzable detergent should be selected.

If pretreatment with enzymes or detergents cannot be used, the freeze / thaw method

should be considered. The freeze / thaw method is commonly used to lyse bacterial

and mammalian cells. The technique involves freezing a cell suspension for 10

minutes in a dry ice and isopropanol bath and then thawing the material at room

temperature immediately prior to ultrasonic processing. The freeze / thaw cycle

should be repeated three times prior to being subjected to the ultrasonics. This

method of lysis causes cells to swell and ultimately break as ice crystals form during

the freezing process and then contract during thawing. This pretreatment has been

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Sonics VCX750 Bedienungsanleitung Seite 27