Biology Reference
In-Depth Information
10. Incubate samples for 4-16 h at RT (see Note 2) with gentle
vortexing to avoid mechanical disruption of the ECM in the
presence of SDS.
11. The extraction buffer is centrifuged at 16,000 × g and stored at
−20°C for later use. The processing of the decellularized tissue
samples continues with the third extraction step.
12. Extraction buffer 3 (4 M guanidine-HCl) : Strongly denatur-
ing. 4 M guanidine is very effective in solubilizing most of the
heavily cross-linked, aggregated ECM components, including
large proteoglycans (versican, aggrecan, etc.), small proteoglycans
(decorin, biglycan, etc.), cell attachment matrix glycoproteins
(such as type VI collagen, fi bronectins, and laminins), and
basement membrane components (perlecan, type IV collagen,
etc.). Guanidine induces disaggregation of ECM components
by destabilizing their ionic, disulfide-dependent protein
conformation (
14 ). 4 M guanidine only partially extracts
interstitial collagen. The buffer-to-tissue weight ratio is kept
constant at 10:1.
13. Incubate samples in extraction buffer 3 for 48 h at RT and
vortex vigorously to facilitate the mechanical disruption of the
ECM and the extraction and solubilization of ECM proteins.
14. Remove the supernatant after 48 h, centrifuge at 16,000 × g
and either store the extracts at −80°C for later use (4 M guani-
dine does not freeze at −20°C) or proceed with the removal of
4 M guanidine (recommended, see Subheading 3.3 ).
15. Interstitial collagen extraction (1 M acetic acid and pepsin) :
Wash tissue samples with ddH 2 O three times to remove any
remaining guanidine remnants.
16.
Add 1 M interstitial collagen extraction buffer and incubate sam-
ples at RT for 48-72 h with vigorous vortexing (see Note 3).
17. After incubation, vacuum dry the extraction buffer to remove
the acetic acid. The resulting collagen-rich pellet can be redis-
solved in gel loading buffer.
1.
For removal of 4 M guanidine from the tissue extracts (see
Note 4), mix the extraction buffer with 100% ethanol at −20°C
for 16 h at an ethanol-to-tissue extraction buffer volume ratio
of 5:1.
3.3. Removal of 4 M
Guanidine
2.
Precipitate proteins by centrifugation (16,000 × g for 45 min).
3.
Wash pellets with 90% ethanol and air dry.
3.4. Deglycosylation
Step ( see Note 5)
1.
For the 0.5 M NaCl extracts, add 4× deglycosylation buffer to
a fi nal concentration of 1×.
2.
For the 4 M guanidine extracts, dissolve the precipitated
protein pellets in 1× deglycosylation buffer (see Note 4).
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