Biology Reference
In-Depth Information
Stacking gel buffer (4
)
0.5 M Tris/HCl, pH 6.8
0.4% SDS (Sigma #L5750)
Acrylamide/bis-acrylamide stock solution (74:1)
40% acrylamide solution (Bio-Rad #161-0140) supplemented with 0.54%
bis-acryl amide (w/v) powder (Bio-Rad #161-0210).
Running buffer
50 mM Tris/HCl
384 mM glycine
0.1% SDS (Sigma #L5750) in deionized water
Transfer buffer
50 mM Tris/HCl
40 mM glycine
0.4 mM SDS
in deionized water
20% (v/v) ethanol
Ponceau S staining solution
0.2% (w/v) Ponceau S in 1% acetic acid
CONCLUDING REMARKS
Here, we have summarized our experiences in the detection of tubulin PTMs using
immunocytochemistry and immunoblot. We have described appropriate methods to
detect changes in MT PTMs using a battery of modification-specific antibodies and
suggest several experimental precautions to take in order to avoid misleading inter-
pretations. Because many functional aspects of MT PTMs might be related to gradual
changes rather than to abrupt changes, careful evaluation of changes in MT modifi-
cation patterns could be essential for the understanding of their roles in cells.
Acknowledgments
This work was supported by the Institut Curie, the CNRS, the INSERM, the Labex CelTisPhy-
Bio 11-LBX-0038, the French National Research Agency (ANR) awards 08-JCJC-0007, the
Human Frontier Science (HFSP) grants RGP0023/2008, the Institut National du Cancer
(INCA) grant 2009-1-PL BIO-12-IC-1, and an EMBO Young Investigator Program grant
to C. J.
We thank C. Benstaali, P. Marques (Institut Curie, Orsay, France), A. Fleury-Aubusson
(CBM, Gif-sur-Yvette, France) for technical assistance, and R. Basto, G. Raposo, A. Wehenkel
(Institut Curie), C. Antony (EMBL Heidelberg), and N. Spassky (ENS, Paris, France) for in-
structive discussions. We are grateful to J.M. Andreu (CSIC, Madrid, Spain) for the kind gift of
the antibody C105. The monoclonal antibody 12G10 developed by J. Frankel and M. Nelson
was obtained from the Developmental Studies Hybridoma Bank developed under the auspices
of the NICHD and maintained by the University of Iowa.
The authors declare no competing financial interests.
