Biology Reference
In-Depth Information
pieces. Proteins will not be extracted effi ciently from large specimen.
Frozen samples should be defrosted but always kept on ice until
dissection and weighing are complete. Cardiovascular samples are
invariably contaminated with plasma proteins. To decrease this
contamination, place samples in 10 mL of 1× PBS, 25 mM EDTA
plus proteinase inhibitors. Mix gently and place on a rotating plat-
form for a few minutes. Change the solution and repeat the proce-
dure fi ve times.
1.
Extraction buffer 1 (0.5 M NaCl): Nondenaturing, ionic
. The
salt ions induce displacement of ionic interactions between
proteins, thus enabling the extraction of weakly bound pro-
teins or protein fragments present in the extracellular space.
These include newly synthesized ECM proteins, which are not
yet cross-linked with the mature ECM, proteolytic fragments
as well as plasma proteins such as lipoproteins, enzymes, and
coagulation factors (
10
).
2. Place samples in 2-mL tubes with a screw cap to avoid leakage.
3.
3.2. Protein Extraction
Add extraction buffer 1. The buffer volume is adjusted to the
weight of the tissue at a ratio of 10:1, i.e., 100 mg of tissue are
incubated in 1 mL of buffer.
4.
Gently vortex samples for 1 h at room temperature (RT). The
optimal incubation time has to be determined experimentally
for each type of tissue (see Note 1).
5.
After incubation, remove the buffer and centrifuge at 16,000 ×
g
.
Freeze supernatant for later use or proceed with deglycosyla-
tion step (see Subheading
3.4
).
6.
Rinse the tissue samples once with ddH
2
O supplemented with
EDTA and proteinase inhibitors before proceeding with the
second extraction step.
7.
Extraction buffer 2 (0.08% SDS): Decellularization.
SDS has
been previously shown to be very effective in solubilizing cyto-
plasmic and nuclear membranes, thereby selectively retrieving
cellular proteins from tissues (
11
). Unlike other decellularizing
agents, such as nonionic detergents (Triton X-100) (
12
), SDS
preserves the ECM and its associated proteins. However,
SDS can cause protein denaturation and disrupt native ECM
proteins (
13
) if used above its critical micelle concentration,
which is 0.24% weight per volume or 8 mM, in water.
8.
Dilute 1% SDS to 0.08% using ddH
2
O and add EDTA and
proteinase inhibitors. There is no need to adjust the pH of the
SDS buffer.
9.
Add the 0.08% SDS buffer to tissue samples at a volume-to-
tissue weight ratio of 10:1.
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