Biology Reference
In-Depth Information
gents are tolerated at concentrations not exceeding 0.5 % (wt/vol);
urea is also tolerated at a concentration not exceeding 3 M.
The method can be applied to a large variety of plant protein
extracts after appropriate elimination of interfering biopolymers.
Nevertheless specifi c aspects of optimization might have to be
considered according to the encountered issues. If for instance
the protein concentration is below 0.1 mg/mL it may be useful
to have a preliminary concentration. This would improve the
capture by CPLL in case the affi nity is too modest. Among pos-
sible concentration methods are dialysis followed by lyophiliza-
tion or membrane concentration under centrifugation.
The presence of proteases, relatively frequent in plant
extracts, is deleterious for the integrity of proteins. Their activ-
ity must be stopped with various selected inhibitors or inactiva-
tion agents prior to contact with CPLL.
3. It may happen that during the capture stage there is formation
of bead aggregates. In this case the supernatant must be sepa-
rated by high-speed centrifugation and the collected solid
material is to be washed extensively with PBS under strong
shaking (e.g., vortex) up to the dissociation of beads from each
other. Chemical agents are not recommended since they may
desorb the captured proteins.
4. The presence of large amounts of high-abundance proteins
after CPLL treatment and protein recovery may mean that the
amount of proteins in the initial sample was not suffi cient to
saturate the beads. This could be resolved by either increasing
the amount of sample or decreasing the volume of CPLL
beads. It is reminded that the enrichment of low-abundance
proteins renders the sample to analyze more complex (many
more proteins are detectable). The subsequent analytical oper-
ations might become of diffi cult interpretation. To facilitate
the analysis it is advised to fractionate the collected proteins or
to elute the capture proteins sequentially.
5. Generally CPLL treatments are highly reproducible; however,
if it is observed that the results are not exactly the same from
an experiment to another, it is advised to check the ionic
strength and the pH of the initial sample. Actually even mod-
est modifi cations of these parameters alter the affi nity of pro-
teins for the peptide baits of the CPLL with consequent
modifi cation of the molecular interaction process.
5
Practical Examples of CPLL-Treated Plant-Derived Extracts
A number of applications have been described in the recent past
with interesting data. To illustrate the power of the described tech-
nology, few examples are reported with particular emphasis on the
sample treatment and the mode of use of CPLL.
