Biology Reference
In-Depth Information
Table 17.1
Advantages and Disadvantages of Different Transfection Methods for
HEK293T Cells
Transfection
Method
Advantages
Disadvantages
Low reproducibility
a
Ca
3
(PO
4
)
2
Inexpensive
Easy to use
Low reproducibility
a
Cytotoxicity at high PEI conc. (
PEI
High expression level
of protein
Inexpensive
Easy to use
10 mg/ml)
>
(
Dong et al., 2007
)
Lipofection
Very high efficiency
Easy to use
Reproducible
Expensive
Not compatible with serum
a
Commercially available analogs often give a better reproducibility.
membrane proteins and cytoplasmic proteins will take place. The pellet contains
the membrane proteins, while the supernatant consists of the cytoplasmic proteins.
An alternative mechanical disruption method when no sonicator is available uses a
polytron. Cells are lysed by homogenization in 7 ml 1
buffer (500 mM Tris, 50 mM
KCl, 10 mM EDTA, 15 mM CaCl
2
6H
2
O, pH 7.4) with a poly-
tron (two times 10 s with incubation on ice in between) followed by centrifugation at
42,000
2H
2
O, 40 mMMgCl
2
g
for 20 min. The pellet contains the membrane fractions and the membrane
proteins can be further extracted and solubilized, using a detergent-based method (see
further). Membrane proteins can also be extracted directly from the cell pellet, without
this homogenization step (see next paragraph). However, in our hands, this results in a
less clear band resolution upon Western blot immunodetection.
17.2.2.2
Nondenaturing detergent-based lysis methods
There are a lot of possibilities concerning detergent choice for the solubilization of
GPCRs (
Allen, Ribeiro, Horuk, & Handel, 2009; Berger, Garcia, Lenhoff, Kaler, &
Robinson, 2005; Corin et al., 2011; Grisshammer, 2009; Nekrasova, Sosinskaya,
Natochin, Lancet, & Gat, 1996; Ren et al., 2009; Seddon, Curnow, & Booth,
2004
), used in the different lysis buffers (
Table 17.2
), for example, CHAPS buffer
(30 mM Tris/HCl pH 7.5; 150 mM NaCl; 1% CHAPS), TNT buffer (20 mM Tris/
HCl pH 7.5; 200 mM NaCl; 0.1% Tween-20), NP-40 buffer (0.5% NP-40;
140 mM NaCl; 1.5 mM MgCl
2
; 10 mM Tris/HCl pH 7.5), and Triton X-100 buffer
(10% glycerol; 1% Triton X-100; 160 mM NaCl; 50 mM Tris/HCl pH 7.5; 1 mM
EDTA; 1 mM EGTA). To prevent degradation or modification of the extracted
proteins by endogenous proteases and phosphatases, specific inhibitors (10
m
g/ml
leupeptin, 1 mM PEFA-block, 2.5
m
g/ml aprotinin, and 10 mM
b
-glycerol phos-
phate) are added to the buffer. Alternatively an inhibitor cocktail can be added
(
Table 17.3
).
Figure 17.1
gives an overview of the extraction of the dopamine D
4
