changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
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3.2.9.6. ZipTip<br />
Day Three: Extraction of Peptides for Analysis (cont.)<br />
The supernatant was carefully collected and comb<strong>in</strong>ed the extract <strong>in</strong><br />
the 0.5 ml plastic micro centrifuge tube.<br />
Step 22 was repeated<br />
Step 23 was repeated<br />
I was followed by carefully collection of supernatant and comb<strong>in</strong>ation<br />
of the extract <strong>in</strong> the 0.5 ml plastic micro centrifuge tube.<br />
The volume of the extract was reduced to less than 20 μL by<br />
evaporation <strong>in</strong> avacuum centrifuge at ambient temperature. The<br />
extract must not be allowed to dry completely.<br />
The volume of the digest was adjusted to ~20 μL with acetic acid.<br />
F<strong>in</strong>ally, the sample was ready for mass spectrometric analysis.<br />
ZipTip is a small C-18 colon used for remov<strong>in</strong>g salts and detergents to obta<strong>in</strong> a<br />
better mass signal. Protocol of ZipTip can be summarized <strong>in</strong> this manner:<br />
ZipTip<br />
1 Wett<strong>in</strong>g 10 μl %100 ACN (Acetonitrile) 10 times<br />
2 Equilibration 10 μl %0.1 TFA (Trifluoroacetic acid) 10 times<br />
3 Sample 10 μl Our Prote<strong>in</strong> Samples 10-15 times<br />
4 Wash<strong>in</strong>g 10 μl %0.1 TFA 5-6 times<br />
5 Elution 5 μl %0.1 TFA/%50 ACN 2-3 times<br />
3.2.9.7. Prote<strong>in</strong> Identification and Mass Spectrometric Analysis<br />
After <strong>in</strong>-gel digestion procedure, two samples (Bortezomib and Control group)<br />
were identified by MALDI-TOF-TOF Mass Spectrometry. The sequence of the<br />
diffentially expressed prote<strong>in</strong> spots were found by us<strong>in</strong>g NCBInr (National Center for<br />
Biotechnology Information, Bethesda, USA) and SwissProt prote<strong>in</strong> databases.<br />
For mass analysis, α-cyano-4-hydroxyc<strong>in</strong>namic acid (HCCA) was used as a<br />
matrix. Its preparationwas described <strong>in</strong> Appendix B.<br />
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