DIGE Analysis of Plant Tissue Proteomes Using a Phenolic Protein Extraction Method - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 340

4. Notes

1. In step 1 of the protocol in Subheading 3.2 , a lysis buffer without
DTT addition is used. At step 5, a lysis buffer containing
the twofold amount of DTT is used. Since the concentration
of DTT in the lysis buffer is variable (20-100 mM), addition of
7.5 mg DTT per mL is adequate at this step.

2. Protein amount can extremely vary in different plant tissues.

Thus, the amount of tissue used for sample preparation has to
be adjusted: In our hands, proteins were extracted from 500 mg
Medicago root tissue ( 20 ), 200 mg Vigna leaf tissue ( 22 ), and
80 mg Cyclamen seeds ( 4 ). A thoroughly prepared sample
obtained by grinding at low temperature (<4°C) improves the
quality of protein extraction. The use of a bead mill (e.g., Retsch
MM 400, Retsch, Haan, Germany) for the pulverization of
plant material is recommended. However, grinding time and
volume of grinding tubes have to be optimized for specifi c plant
material. Low amounts of soft plant material, like leaves, roots,
and embryos, require short grinding times (1-5 min) and can
be performed directly in Eppendorf vessels using steal balls.
Large amounts of hard tissue may be grinded for longer time
periods (up to 30 min). Long storage periods of frozen plant
samples even at −80°C conditions before protein preparation
minimize the yield of extracted total protein.
3. The 2-mL Eppendorf vessels should be fi lled up to three-quarter
with fi ne ground plant tissue powder. When adding the extrac-
tion buffer and subsequently the phenol, the volume of the
sample decreases.

4. All work with -mercaptoethanol should take place under the

extractor hood!

β

5. The quality of the phenol is of high importance. Phenol of

insuffi cient quality can cause dramatic losses in protein yield.
When using a two-phase phenol, it is important to take up the
liquid phase only from the lower phenol phase. Do not shake
the phenol in order to mix both phases. Work with phenols
should only take place under the extractor hood!

6. After centrifugation (step 3), normally, an aqueous phase is

obtained at the bottom of the Eppendorf vessels and a pheno-
lic phase at the top. Plant cell walls and membrane compounds
precipitate at the interface. In rare cases, aqueous and phenol
phases are inverted after centrifugation. In this situation, the
upper phase should be carefully removed with a pipette before
collecting the phenol phase. Generally, transfer the phenol
phase in several small volume steps (e.g., in 2-6 steps each
collecting 100 μ L).