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In-Depth Information
5. Total proteins were separated in the fi rst dimension in different
pH fractions of 0.3 range. The total number of fractions
obtained was 34 and 15 fractions were selected for the second
dimension using the HPRP chromatography. In Fig.
2b
the
855 bands obtained in the 15 fractions used for the second
dimension are represented. A virtual 2D gel was given by the
software, combining the fi rst and the second dimension, thus
showing proteins separated by pI and hydrophobicity.
6. We have analyzed the protein resolution at different pH ranges
and found that the differences were signifi cant (Fig.
2
).
Despite the advantage of using the ProteomeLab PF 2D for
analyzing protein expression with high loading capacity that
enhances the detection of low-abundance proteins with few
purifi cation steps, thus achieving a higher level of reproduc-
ibility than the chemical 2DE, the resolution of proteins
depended on the pH gradient.
7. Liquid chromatography separates proteins uniformly over a
pH gradient and 2DE methods follow a normal trend in
protein distribution over the same pH gradient. This means
that liquid chromatography is better in the resolution of
proteins at extreme pH (over 7.0 and below 4.5) while 2DE
provides a higher resolution at pH 5-6.5 (Fig.
3
).
Acknowledgment
This work was supported by Bio 2009-13044-Co2-01 from
MICIN.
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