Biology Reference
In-Depth Information
Table 17.2
Types of Detergents and their Characteristics
Type of Detergent
Description of Use
Example
Ionic detergent
Very effective in the solubilization of membrane
proteins but denaturing the proteins to some
extent
SDS
Bile acid salts
Mild detergent and less inactivating
doc
Nonionic detergent
Mild surfactants that do not break protein-protein
interactions and do not denature proteins
Triton
X-100
Tween-20
DDM
NP-40
Zwitterionic
detergent
More harsh surfactants than the nonionic but
conformation and interactions are preserved
CHAPS
Table 17.3
Function of the Specific Inhibitors
Inhibitor
Function
Leupeptin
Protease inhibitor; can inhibit cysteine, serine, and threonine
peptidases
PEFA-block
Serine protease inhibitor
Aprotinin
Trypsin inhibitor
b-Glycerol phosphate
Serine and threonine phosphatase inhibitor
Protease inhibitor
cocktail
Inhibits serine, cysteine, aspartic, and metalloproteases
receptor from transiently transfected HEK293 cells with different lysis methods.
The conclusion for the dopamine D
4
receptor is that a RIPA buffer seemed to be
the optimal choice, because it gave the best signal on SDS-PAGE (
Fig. 17.1
). For
this reason, a detailed protocol for the RIPA lysis is provided in the following
section.
17.2.2.2.1
Radioimmunoprecipitation (RIPA) lysis
The membrane fractions are resuspended in 300-500
m
l of RIPA buffer (150 mM
NaCl; 50 mM Tris/HCl pH 7.5; 1% NP-40; 0.1% SDS; 0.5% deoxycholic acid) with
freshly added inhibitors (
Table 17.3
) for 1-2 h at 4
C. This lysis buffer is based on
moderate concentrations of the nonionic detergent NP-40 that will disrupt the integ-
rity of the cell membrane, causing the extraction of membrane proteins in their native
form. The buffer also contains a low concentration of the harsh denaturing anionic
detergent SDS and ionic detergent sodium deoxycholate (doc). SDS and doc will
help to disrupt the membrane, resulting in a higher yield of extracted membrane
proteins. The concentration of these two is however quite low and will not be able
to disrupt the protein-protein interactions. In addition, the detergent digitonin (0.1%)
