Symbiotic Fungi: Principles and Practice (Soil Biology)

15 Functional Genomic of Arbuscular Mycorrhizal Symbiosis 247
Table 15.1 Extraction buffer (100 ml) for total and soluble proteins
Tris 0.5 M 6.055 g
Milli-Q water Adjust the pH to 7.5 and
complete to 50 ml
Sucrose 0.7 M 23.900 g
KCl 0.1 M 0.750 g
Thiourea 10 mM 0.076 g
EDTA 5 mM 50 mM 10 ml
b-mercaptoethanol 2% a
2ml
PMSF 1 mM in DMSO 0.017 g. DMSO ml 1
Milli Q water
a
To be added just before using the solution
sqf 100 ml
Table 15.2 Solubilization buffer for soluble proteins a
Urea 9 M 5.4 g
CHAPS 4% (w/v) 0.4 g
Triton X-100 0.5% (v/v) 50 ml
DTT 100 mM 0.154 g
IPG buffer 3–10 2% (w/v) 200 ml
Milli Q water sqf 10 ml
a Can be stored at 20 C
Cold methanol and acetone
Solubilizing buffer (see Table 15.2) [modification of O’Farrell lysis buffer
(O’Farrell, 1975)]
Liquid nitrogen N2
Gas nitrogen
15.3.2 Products and Buffers for 2DE
Note 1: All chemical for electrophoresis must be high quality grade IPG
strips (ready-made IPG gels Immobiline) such as Immobiline DryStrips 3–10 NL,
Immobiline DryStrips 4–7 (or any other restricted gradient)
IPG buffers in the same gradient range as IPGs
Mineral oil (Sigma)
Acrylamide
Agarose
Urea
SDS
Iodoacetamide
TEMED
Ammonium persulfate