Biology Reference
In-Depth Information
8. Remove the supernatant with a pipette to avoid losing the
pellet. Be careful: do not move the pellet because it is very easy
to disintegrate
9. Remove the supernatant with a pipette to avoid disaggregating
the pellet and carry contaminant.
10. The pellet of the albumins fraction appearance may be viscous;
this aspect is normal, indicating that it may contain the remain-
ders of lipids and polysaccharides [
29
].
11. Dry the pellet to ensure that the acetone was completely
removed, if it lasts for a long time is diffi cult to resuspend the
protein.
12. The pellet of the globulins fraction has two phases, namely,
one solid and another oily. Remove the last one and use the
solid phase to resuspend proteins.
13. The glutelins solubilization is performed at room temperature
to prevent the insolubilization of SDS.
14.
Steps 1
-
3
of each fraction solubilization should be repeated in
order to make sure that the maximum amount of proteins cor-
responding to each fraction are solubilized.
15. The volume of buffer to resuspend proteins depends on the
amount of precipitate obtained. It is recommended that sam-
ples are well concentrated.
16. The insoluble material is abundant in the case of the glutelin
fraction.
17. The extracts are cleaned in this step, removing contaminants
that may interfere with the identifi cation by MS such a salt,
detergents, etc.
18. For more details you can consult the following review [
30
].
Acknowledgments
This work has been fi nanced by the Spanish Ministry of Innovation
and Science, project AGL2009-12243-C02-02 “Variability, cata-
loguing, responses to stresses and clonal propagation of holm oak
(
Quercus ilex
subsp.
ballota
) (DECOVA)” (FEDER Co-fi nanced)
and the Andalusian Government (Junta de Andalucía). MC
Romero Rodríguez has been supported by a grant from the
“Fundación Parque Tecnológico Itaipu-Binacional” and “Centro
Multidisciplinario de Investigaciones Tecnológicas - Dirección
General de Investigación Científi ca y Tecnológica-Universidad
Nacional de Asunción - Paraguay”.
