Biology Reference
In-Depth Information
Peroxidases with different
glycosylation patterns
Chitinases
Thaumatin-like proteins
Fig. 1 SDS-PAGE followed by silver protein staining of the elicited extracellular
medium of V. vinifera. Lane M , Molecular weight markers; Lanes 1-3 , extracellular
medium obtained from treated grapevine cells with methyl
-cyclodextrin
(MBCD) alone ( lane 3 ) or in combination with methyl jasmonate (MeJA) at different
concentrations (450
β
μ
M, lane 2 and 270
μ
M, lane 1 )
2. For Coomassie brilliant blue staining ( see Note 23 ), place the
gel in a square Petri dish containing 100 mL Coomassie stain-
ing solution. Incubate the gel for 45 min to 3 h in the staining
solution. Destain the gel by adding 100 mL Coomassie destain-
ing solution. To remove the background, destain the gel with
several changes of Coomassie destaining solution.
3. Keep gels in distilled water for short-term preservation. It
might be convenient to transfer carefully the gel to a heat-
sealable bag for longer term storage.
4. Excise the bands of interest from gel, and place them in dif-
ferent tubes containing distilled water until their
processing.
3.6 In-Gel Protein
Digestion
Once the interest bands are cut from gels, deposit in 96-well plates
and process automatically in a Proteineer DP (Bruker Daltonics).
The digestion protocol used is based on that described by
Schevchenko et al. [ 33 ] with minor modifi cations. Wash gel plugs
with 25 mM ammonium bicarbonate to remove dye and SDS
impurities. Reduce the samples with 60 mM DTT and alkylate cys-
teines by adding an excess of iodoacetamide followed by digestion
with porcine trypsin (Promega) at 37 °C for 6 h. Extract peptides
by washing in 25 mM ammonium bicarbonate, then in 70 % ACN,
and then in 1 % FA. Finally dry tryptic peptides using a speed-
vacuum centrifuge.
3.7 LC-MS/MS
Identifi cation and
Database Searches
1. Resuspend tryptic peptides obtained from in-gel digestion
(Subheading 3.6 ) in 100
μ
L of 0.1 % FA and 3 % ACN, suitable
for LC-MS/MS analysis.
2. Concentrate and desalt the tryptic peptides on a Zorbax
300SB-C18 cartridge and separate on an analytical Zorbax RP
C18 column with an Agilent 1200 HPLC system.
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