Biology Reference
In-Depth Information
Anti-detyr-tubulin rabbit polyclonal antibody (Millipore #AB3201;
Paturle-
Lafanechere et al., 1994
), raised against the -CGEEEGEE(-COOH) peptide, recog-
nizes the C-terminal -GEE sequence of detyrosinated
a
-tubulin. Another antibody,
the mouse monoclonal 1D5 (Synaptic Systems #302011;
R¨diger et al., 1999; Warn
et al., 1990
), also detects C-terminal stretches of two and more glutamate residues
(
2). However, while 1D5 detects detyrosinated
a
-tubulin (which ends with
C-terminal -EE in most species), it cross-reacts with any polyglutamylated form of
tubulin that contains glutamate side chains that contain two or more glutamate res-
idues. Thus, the use of this antibody alone makes the interpretation of the data
impossible.
△
E
n
;
n
2-tubulin (see
Box 16.1
) can be detected by a rabbit polyclonal antibody
(Millipore #AB3203;
Paturle-Lafanechere et al., 1994
) that has been raised against
the peptide -CEGEEEGE(-COOH). Anti-
2-tubulin antibody detects specifically
C-terminal -GE sequence of proteins (
Rogowski et al., 2010
).
△
16.2.1.3
Polyglutamylation
Twoantibodies allowassessing the extent of both
a
-and
b
-tubulinpolyglutamylation: the
mousemonoclonalGT335 (Adipogen #AG-20B-0020;
Wolff et al., 1992
), which recog-
nizes the epitope formed at the branching point of the glutamate side chains (
g
-linked
glutamate on a main-chain glutamate), and thus stains both short and long side chains
of tubulin polyglutamylation. It has been raised against the branched peptide
-EGEGE*EEG(-CONH
2
) with a bi-glutamate side chain (-EE(-COOH)) attached to
the
g
-carboxygroupof theglutamatemarkedwiththeasterisk.GT335alsodetectsa range
of nontubulin substrates of glutamylation (
Regnardetal.,2000;vanDijketal.,2008
).
Polyglutamylation can be detected using the rabbit polyclonal antibody polyE
(
Rogowski et al., 2010; Shang et al., 2002; van Dijk et al., 2007
). This antibody is still
not commercially available. It detects stretches of at least three glutamate residues situ-
atedat theC-terminal extremity of proteins andhas been raisedagainst a -CEEEEEEEEE
(-COOH) peptide, and is therefore specific to long side chains added by polyglutamyla-
tion to tubulin and other substrates, and it also detects proteins that have a genetically
encoded C-terminal tail with three or more glutamate residues (
Rogowski et al., 2010
).
The use of these two antibodies, GT335 and polyE, allows distinguishing between
long (GT335- and polyE-positive) and short (GT335-positive, polyE-negative) side
chains generated by polyglutamylation.
16.2.1.4
Polyglycylation
The mouse monoclonal antibody TAP952 detects monoglycylated tubulin (
Callen
et al., 1994
), but unlike GT335 (detecting both short and long glutamate chains),
it does not cross-react with elongated glycine side chains. TAP952 has been raised
against Paramecium axonemal tubulin and been shown to detect
g
-linked glycine
residues on glutamic acids 437, 438, 439, and 441 of peptides corresponding to
C-terminal tails of paramecium
b
-tubulin (
Br´, Redeker, Vinh, Rossier, &
Levilliers, 1998
). Though the antibody has been shown to detect strongly glycylated
tubulin in cilia and flagella of many species, it is not yet clear whether all possible
