Biology Reference
In-Depth Information
4
Notes
1. Mega GeBAfl ex-tubes were used for dialysis to reduce loss of
material. Different tube caps can be used depending on the
volume to dialyze (3-20 mL).
2. Due to their hygroscopic nature, MgCl
2
and MnCl
2
salts must
be kept in closed bottles in a dry atmosphere.
3. Buffer B1 should be prepared from stock solutions because
MnCl
2
might precipitate when powders are introduced all
together.
4. ConA agarose resin is supplied as a suspension. Take a twofold
volume of this suspension to get the volume required for
chromatography.
5. When preparing the SDS solution, warm it in a 60 °C water
bath to facilitate SDS dissolution. If precipitation occurs during
storage, the same procedure should be carried out.
6. APS and TEMED being polyacrylamide polymerizing agents,
they need to be introduced last during resolving and stacking
gel preparation. Gels should be poured immediately.
7. Prevent drying of the soil. It should remain wet.
8. Avoid xylem sap harvesting during the summer. Plants can be
stressed by high temperatures, especially cabbages. The quantity
of sap collected is quite low.
9. Room temperature should be 20-25 °C.
10. Avoid placing the mini-greenhouses in ventilated areas.
11. The cut should be clean.
12. Collection takes place every 30 min to 1.5 h. Monitor and
collect the drops before they fall of the cut surface. Quickly
close the mini-greenhouses after each sampling to prevent
drying of the sections.
13. Total hexose analysis suggests that the two fi rst fractions are
probably contaminated with phloem sap or cytoplasm of cut
cells (T Dugé de Bernonville, unpublished data).
14. Protein content in the xylem sap protein extract can be too low
to be quantifi ed using the Bradford method. The same prob-
lem can be encountered after ConA chromatography. However,
protein detection is possible after protein separation by SDS-
PAGE and staining with colloidal blue or silver nitrate.
15. ConA lectin affi nity chromatography is specifi c for Man residues
and allows specifi c capture of
N
-glycoproteins [
34
]. This
approach can be carried out for xylem sap proteomics since
most proteins are
N
-glycosylated through the secretion pathway.
It allows enrichment in minor proteins and consequently
enlargement of the proteome coverage.
