Biology Reference
In-Depth Information
CHAPTER
16
Investigating Tubulin
Posttranslational
Modifications with Specific
Antibodies
Maria M. Magiera and Carsten Janke
Institut Curie, CNRS UMR3306, INSERM U1005, Orsay, France
CHAPTER OUTLINE
Introduction ............................................................................................................ 248
16.1 Observations...................................................................................................249
16.1.1 Impact of Cell Fixation on Antibody Labeling ................................ 249
16.1.2 The Importance of Antibody Concentration in Analyzing MT PTMs in
Cells ......................................................................................... 252
16.1.3 Antibody and Sample Concentration in Immunoblot Analysis.......... 253
16.2 Materials and Methods....................................................................................255
16.2.1 Antibodies Detecting Tubulin PTMs ............................................. 255
16.2.1.1 Acetylation......................................................................... 255
16.2.1.2 Tyrosination/detyrosination/
2-tubulin ................................ 256
16.2.1.3 Polyglutamylation............................................................... 258
16.2.1.4 Polyglycylation ................................................................... 258
16.2.2 Cell Culture and Fixation............................................................. 259
16.2.2.1 Cell Culture and Transfection ............................................. 259
16.2.2.2 Cold Methanol Fixation ...................................................... 259
16.2.2.3 PFA Fixation...................................................................... 259
16.2.2.4 DSP-PFA Fixation.............................................................. 259
16.2.3 Immunocytochemistry on Fixed Cells ........................................... 260
16.2.4 Microscopy and Image Acquisition ............................................... 260
16.2.5 Electrophoresis and Immunoblot.................................................. 260
16.3 Buffer Composition..........................................................................................261
16.3.1 Cell Fixation............................................................................... 261
16.3.1.1 PFA Method ...................................................................... 261
16.3.1.2 DSP-PFA Method.............................................................. 261
16.3.2 Gel Electrophoresis and Immunoblotting ...................................... 262
16.3.2.1 SDS-PAGE for a/b-tubulin Separation ................................. 262
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