Biology Reference
In-Depth Information
Cotyledons and
embryo grinding (liquid N
2
)
Pericarp removal
Diethylether washes
Addition of DTT
Lipid removal
Un-lipped homogenated pellet
ES 1
Tris-HCl, DTT,
MgCl
2
, CaCl
2
, PVPP
Water-soluble
fraction
Albumins
In gel cleanup
Pellet 1
ES 2
Tris-HCl, NaCl, DTT,
EDTA, EGTA, PVPP
MS ANALYSIS
Dilute salt-soluble
fraction
Globulins
LC-MS in tandem (LTQ-Orbitrap)
Pellet 2
Alcohol-water
mixture-
soluble
fraction
ES 3
75% ethanol (v/v),
PVPP
Data base
searching
Prolamins
Pellet 3
Protein
identification
ES 4
Sodium borate,DTT,
SDS, PVPP
Acid & alcali-
soluble
fraction
Glutelins
Pellet 4
Fig. 1
Flowchart of the protein extraction procedure and MS analysis
2
Materials
1. Analytical Mill, Model A-10 (115 V), Janke & Kunkel IKA
Labortechnik.
2. Mortar, pestle, and knife.
3.
Ultrasonic Homogenizer 4710 series
; Cole-Parmer Instrument Co.
4. Refrigerated centrifuge Model J2-21; Beckman.
5. Microcentrifuge Model 5415 D, Eppendorf.
6. BioPhotometer, Model 22331, Eppendorf.
7. Vertical Electrophoresis equipment. Mini PROTEAN II, Bio
Rad.
8. Power supply, PowerPac HC, Bio-Rad
2.1 Equipment
( See Note 1 )
