Biology Reference
In-Depth Information
identifi ed. A total of 224 of these identifi ed proteins were also
quantifi ed, taking into account only those with at least 3 detected
peptides [
59
].
Summarizing, we have shown in this chapter several protocols
to work with different fungal material based on our experience
with the phytopathogenic fungus
B. cinerea
. Moreover, the use of
complementary proteomic approaches, as gel-based (1-DE and
2-DE) and gel-free (label-free LC-based) techniques, provides
higher proteome coverage, relevant information being obtained
on fungal biology and their interaction with their hosts [
3
,
4
,
12
,
45
,
59
].
5
Notes
1. Gloves should be used for these procedures, and special care
should be taken when handling TCA and phenol (consult
safety data sheets) since they are corrosive products. Steps
involving phenol and
β
-mercaptoethanol should be performed
in a fume hood.
2. Examples of rich-media are PDAB (potato, dextrose,
agar + bean leaves), solid synthetic complete medium (CM)
[
70
], or solid malt extract medium (1.5 % w/v).
3. Be careful when working with liquid nitrogen: due to its cool
temperature (−195.8 °C) it may cause severe frostbite. The
nitrogen evaporates reducing the concentration of oxygen in
the air and may act as an asphyxiating agent, especially in con-
fi ned spaces. Remember that it may be dangerous because
nitrogen is odorless, colorless, and tasteless, and it could cause
suffocation without any sensation or warning.
4. Be careful: do not throw out the pellet.
5. Three phases appear, namely, the upper phase (which is the phe-
nolic phase where proteins are), a white interphase, and a lower
aqueous phase. Try not to get parts from the white interphase.
6. The volume of solubilization solution added will depend on
the quantity of precipitated proteins. It is advisable that sam-
ples be well concentrated.
7. In this case, the precipitated pellet may be faint because the pro-
teins secreted to the medium are at a very low concentration.
8. The problem derived from the use of solutions without thio-
urea, CHAPS, and Triton X-100 is the loss of hydrophobic
proteins.
9. Acrylamide gel (5 %) (for 10 mL monomer solution): Mix
5.7 mL of ddH
2
O, 1.7 mL of 30 % (v/v) acrylamide-bisacryl-
amide BioRad mixture, 2.5 mL of 0.5 M Tris-HCl pH 6.8 buffer,
