Biology Reference
In-Depth Information
All the above sample pretreatments contribute to obtain
signifi cantly better analytical results especially when using 2D
electrophoresis and related methods. One of the preferred
methods for protein recovery is the precipitation with ammonium
sulfate at high concentration (80-90 % saturation). Naturally this
salt is to be removed, which is accomplished by simple dialysis at a
very low cutoff (e.g., 3,500 Da) or by centrifugation using appro-
priate fi ltration-integrated devices.
When dealing with vegetable-derived beverages such as wines
no specifi c pretreatment is really necessary.
3.2 Protein Capture
and Dynamic Range
Reduction
Once the plant protein extract is obtained (
see
Note 2
) the treatment
with CPLL is relatively standard. Nevertheless few variations allow
capturing more or less proteins. The general protocol is as follows:
1. Solid-phase hexapeptide ligand libraries whatever the presenta-
tion, dry or wet, need to be equilibrated with the buffer solu-
tion selected for protein capture (e.g., PBS) before use. To this
end 100
L of selected
solution and gently agitated for few hours or overnight. Then
the beads are washed extensively with the same fresh buffer
under mild centrifugation at for example 2,000-3,000 ×
g
.
Finally all excess of solution is removed by centrifugation at
12,000 ×
g
or more.
2. The plant extract in the selected buffer used for protein capture
and containing the antiprotease cocktail is centrifuged at 12,000 ×
g
to remove all solid material in suspension. For an optimal capture
the protein concentration of the sample should be at least of
0.1-1 mg/mL. Lower concentrations may render the capture of
very-low-abundance proteins diffi cult when the dissociation con-
stant is too high. The total amount of protein from the sample
should be larger than 50 mg when 100
μ
L of CPLL are mixed with 500-1,000
μ
L of hexapeptide ligand
library is used. The larger the protein load, the greater the proba-
bility to enrich and thus detect very-low-abundance proteins. To
concentrate a too dilute protein solution current methods adopted
in biochemistry techniques can be used. Preferred ways are lyophi-
lization after dialysis or membrane concentration under centrifuga-
tion (Centricon, centrifugal fi lters from Millipore).
μ
3. The protein solution is mixed with the drained CPLL beads
and the suspension gently agitated for at least 2 h at room tem-
perature (
see
Note 3
). It is critical to maintain an even slurry of
the hexapeptide ligand library beads so as to ensure good con-
tact between the beads and the proteins. The agitation should
not be too strong so as to avoid formation of foam. The suspen-
sion can be left overnight under agitation without negative
consequences. These very important protein capture steps can
be performed in physiological conditions of ionic strength and
pH; however, they can also be made in lower ionic strength to
