Ionic detergents CHAPS

The addition of zwitterionic or non-ionic detergents to SD buffers has three important functions. Detergents aim to disrupt membranes and consequently solubilize lipids and delipidate proteins, which are bound to membranes or vesicles. The use of SDS as a detergent will affect the overall net charge and so non-ionic or zwitterionic detergents such as Triton X-100 or CHAPS are used (Shaw and Riederer, 2003). Commonly used CHAPS, which will not affect the overall charge, can be added up to 4% in SD buffers to account for... 

SDS, sodium dodecyl sulfate 2-ME. 2-mercaptoethanol CHES, cyclo-hexylamine sulfonic acid NP-40, Non-ident 40 non-ionic detergent PMSF, phenylmethylsulfonyl fluoride CHAPS, 3-t(3-cholam1dopropyl) dImethvlammonloH -propane sulfonate. ... 

Alternative protocols are described elsewhere (Ribeiro-Neto efal., 1985 Kopf and Woolkalis, 1991 Carty, 1994). ATP, phospholipids and small amounts of certain ionic and nonionic detergents (i.e. SDS, CHAPS, Lubrol PX) promote dissociation of the A and B protomers (Moss et a/., 1986). We do not use SDS since it denatures solubilized G proteins very easily. DTT or 3-ME are necessary to break the disulfide bonds of the A protomer. Supplementation with BSA (final concentration approx. 0.9 mg/ml) helps prevent loss of enzyme through adsorption to the walls of the tube, and ensures recovery of proteins following precipitation with sodium chloride/acetone, trichloroacetic acid (TCA), or chloroform/methanol. Furthermore, when samples are subjected to SDS-PAGE, intensities of the stained 67 kDa BSA protein bands allow rough estimation of incomplete recovery of the precipitated sample (see section 4.5). Preactivated PT should be used immediately, and enzyme left over from an experiment should be discarded, since reduced toxin has been shown to lose activity rapidly (Kaslow et a/., 1989). 

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