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 ...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
2.1.4.1.2. Two-Dimensional Gel Electrophoresis<br />
(2D PAGE) .......................................................... 32<br />
2.1.4.2. Detection of Prote<strong>in</strong> Spots and Image Analysis ............... 34<br />
2.1.4.3. In-Gel Digestion ............................................................... 35<br />
2.1.4.4. ZipTip (C-18 Colon) ......................................................... 36<br />
2.1.4.5. Mass Spectrometry ........................................................... 37<br />
2.1.4.5.1. Ionization Methods ............................................... 38<br />
2.1.4.5.1.1. Matrix-Assisted Laser<br />
Desorption/Ionization (MALDI) ......... 38<br />
2.1.4.5.2. Mass Analyzers .................................................... 40<br />
2.1.4.5.2.1. Time-of-Flight Mass Analyzer (TOF) 41<br />
2.1.4.5.3. Detectors ............................................................... 43<br />
2.1.4.6. Tandem Mass Spectrometry (MS/MS) ............................. 43<br />
2.1.5. Advantages and Drawbacks of Prote<strong>in</strong> Profil<strong>in</strong>g ........................ 43<br />
2.1.6. Aim of the Study ......................................................................... 45<br />
CHAPTER 3. MATERIALS AND METHODS ............................................................ 46<br />
3.1. Materials ............................................................................................. 46<br />
3.1.1. Medias ......................................................................................... 46<br />
3.1.2. Chemicals, Reagents and Solutions ............................................ 46<br />
3.2. Methods .............................................................................................. 46<br />
3.2.1. Cell L<strong>in</strong>es and Culture Conditions .............................................. 46<br />
3.2.2. Thaw<strong>in</strong>g the Frozen Cells ........................................................... 47<br />
3.2.3. Freeze the Cells ........................................................................... 47<br />
3.2.4. Cell Viability Assay .................................................................... 47<br />
3.2.5. Cell Proliferation Assay .............................................................. 48<br />
3.2.6. Evaluation of Apoptosis .............................................................. 50<br />
3.2.6.1. Measurements of Caspase-3 Enzyme Activity ................. 50<br />
3.2.6.2. Bradford Prote<strong>in</strong> Assay for Prote<strong>in</strong> Determ<strong>in</strong>ation .......... 53<br />
3.2.6.3. Detection of the Loss of Mitochondrial Membrane<br />
Potential (MMP) .............................................................. 56<br />
3.2.7. Total Prote<strong>in</strong> Extraction from MM U-266 Cells ........................ 57<br />
3.2.8. Determ<strong>in</strong>ation of Prote<strong>in</strong> Concentration ..................................... 58<br />
3.2.9. 2D-PAGE Analysis ..................................................................... 58<br />
vii