production of selected secondary metabolites in transformed ...
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Fig.1: Chemical structures <strong>of</strong> pI markers used <strong>in</strong> this work.<br />
Polyacrylamide gels and IEF - The prote<strong>in</strong> samples (2 mg/ml) were mixed with pI markers<br />
(10 mg/ml each) and the 4 μl <strong>of</strong> the mixtures were loaded onto the polyacrylamide gel (16%).<br />
The carrier BioLyte 3/10 ampholyte (f<strong>in</strong>al concentration 2%) (Bio-Rad, CA, USA) were<br />
added prior to gel polymerization. Separation was performed with constant electric power <strong>of</strong><br />
0.6 W for 2 h supplied by VNZ 22 power supply (CSAV Development Workshop, Czech<br />
Republic) us<strong>in</strong>g a model 111 M<strong>in</strong>i IEF Cell (Bio-Rad, CA, USA). After focus<strong>in</strong>g completion<br />
the gels were gently removed from electrodes and scanned.<br />
Ladders <strong>of</strong> color pI markers simplify the orientation <strong>in</strong> the gel even when the separated<br />
prote<strong>in</strong>s cannot be seen. Thus, the bands with prote<strong>in</strong>s were excised accord<strong>in</strong>g to positions <strong>of</strong><br />
pI markers. The excised gel pieces were then transferred to 0.5 ml tubes. The pI markers were<br />
then eluted from gel by 30 μl water/ethanol 1:1 (v/v) for 15 m<strong>in</strong>.<br />
Prote<strong>in</strong> digestion and identification - Excised gel pieces after the extraction <strong>of</strong> the pI markers<br />
were washed twice with water/acetonitrile 1:1 (v/v) for 15 m<strong>in</strong>. Then prote<strong>in</strong>s were identified<br />
accord<strong>in</strong>g to standard proteomic protocol utiliz<strong>in</strong>g <strong>in</strong>-gel digestion <strong>of</strong> reduced and alkylated<br />
prote<strong>in</strong>s with tryps<strong>in</strong>. 2<br />
For comparison, the other IEF focused gels were sta<strong>in</strong>ed with Coomassie Brilliant Blue R<br />
250. Due to the carrier 3/10 ampholytes used for creat<strong>in</strong>g <strong>of</strong> pH gradient <strong>in</strong> gels precipitated<br />
the Coomassie dye dur<strong>in</strong>g sta<strong>in</strong><strong>in</strong>g caus<strong>in</strong>g strong blue haze on the background, the wash<strong>in</strong>g<br />
step (10% TCA <strong>in</strong> water/methanol (10/3 v/v); 12-14 h) had to be <strong>in</strong>serted prior to own<br />
sta<strong>in</strong><strong>in</strong>g procedure.<br />
Mass spectrometry - MS measurements were carried with 4700 Proteomics Analyzer<br />
(Applied Biosystems, USA) MALDI TOF/TOF mass spectrometer (equipped with Nd/YAG<br />
laser; 355 nm). Argon was used as a collision gas. A solution <strong>of</strong> s<strong>in</strong>ap<strong>in</strong>ic acid (SA) (20<br />
mg/ml <strong>in</strong> acetonitrile/water (3/2 v/v)) was used as matrix for markers. Alpha-cyano-4hydroxyc<strong>in</strong>namic<br />
acid (CHCA) (both Sigma-Aldrich, Germany) was used as matrix for<br />
peptides <strong>in</strong> concentration <strong>of</strong> 10 mg/ml acetonitrile/water (3/2 v/v).<br />
Sborník soutěže Studentské tvůrčí č<strong>in</strong>nosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006<br />
Sekce DSP 2006, strana 255