Biology Reference
In-Depth Information
17.2.2.3
Denaturing-based lysis methods: urea lysis and SDS-lysis
Cells are resuspended in 6 M urea lysis buffer (20 mM Tris/HCl pH 7.5; 135 mM
NaCl; 1.5 mMMgCl
2
; 1 mM EGTA; 1% Triton X-100; 10% glycerol and 6 M urea).
The presence of Triton X-100 in the buffer will help with the solubilization of the
membrane proteins. Upon incubation for 30 min at room temperature, the solution
is centrifuged (8000
g
, 5 min), and the supernatant fraction will contain the solu-
bilized membrane proteins. However, these proteins will be completely denatured as
urea is a powerful protein denaturant that disrupts noncovalent bonds.
Another lysis method resulting in solubilization and complete denaturation of
proteins is the SDS-lysis method. With this method, you resuspend the cells in
SDS-lysis buffer: 62.5 mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 0.1% bromo-
phenol blue, and 50 mM dithiothreitol (DTT). The presence of SDS together with the
DTT will result in the denaturation of all the proteins in the sample. Glycerol is added
to facilitate sample loading on SDS-PAGE gel and bromophenol blue is added to
visualize sample separation.
For studying protein-protein interactions, it is extremely important to keep pro-
teins in their native form after lysis. Therefore, both methods, in which proteins are
denatured during lysis procedure, are not useful for co-IP or mco-IP.
17.2.3
Coimmunoprecipitation
Coimmunoprecipitation is a common method used to study protein-protein interac-
tions. In this technique, one of the potential interacting partners (
protein X
) is pre-
cipitated from the solution using an antibody that specifically binds to this protein X.
The antibody can be directed against endogenous epitopes or against an exogenous
tag (e.g., HA, FLAG, and His), added to the receptor with the help of genetic engi-
neering methods. A comparison of advantages and disadvantages of both approaches
can be found in
Table 17.4
. The antibody used for immunoprecipitation needs to be
coupled at some moment to a solid resin. This resin is created by agarose/polyacryl-
amide/Sepharose or magnetic beads to which recombinant Protein A or G has been
Table 17.4
Advantages and Disadvantages of Antibodies Against Endogenous and
Exogenous Epitopes Used for Immunoprecipitation Technique
Antibody
Recognizing
Advantages
Disadvantages
Endogenous
epitope
Isolation of protein from native
tissue
Does not require overexpression
Lack of good antibodies for a lot
of proteins esp. GPCRs
Often much lower efficiency
Exogenous
epitope tag
Very good efficiency
No need of having a separate
antibody for each protein
Availability of beads with
immobilized antibody
Overexpression of protein is
required
No possibility to isolate proteins
from native tissue
