Biology Reference
In-Depth Information
the optimal (or better minimal) concentration (
Fig. 16.3
A), another important factor
is the dilution of the protein samples that are loaded on the SDS-PAGE. Because
tubulin, even at relatively high concentrations, resolves into one (standard SDS-
PAGE) or two (specific SDS-PAGE to resolve
a
- and
b
-tubulin;
Lacroix &
Janke, 2011
and described below) neat bands, overloading of the antigen is often
not noticed. In contrast, differences in tubulin modification levels are easily over-
looked if too much material has been loaded on the SDS-PAGE prior to immunoblot
(
Fig. 16.3
B). As the presence of tubulin PTMs does not significantly alter the appar-
ent molecular weight of tubulin separated on an SDS-PAGE, there is no additional
hint, apart from the antibody detection levels, indicating the PTM state of tubulin.
An additional factor that can easily obscure the need to dilute the antibodies
of tubulin PTMs is that most of these antibodies yield only very low background
signals, even if used in high concentrations. This has been observed for both,
immunocytochemistry and immunoblot (
Fig. 16.3
A), and could thus hamper the
identification of differences in modification levels in these two experimental
setups.
16.2
MATERIALS AND METHODS
16.2.1
Antibodies detecting tubulin PTMs (summarized in
Table 16.1
)
16.2.1.1
Acetylation
So far, only one site of tubulin acetylation has been studied in detail. Acetylation of
lysine-40 of
a
-tubulin is specifically detected with the mouse monoclonal antibody
6-B11-1 (IgG2b isotype), which has been raised against the axonemal
a
-tubulin from
sea urchin sperm flagella (Sigma #T6793, #T7451;
LeDizet & Piperno, 1987, 1991;
subjected to indirect immunofluorescence using C105 (b-tubulin) to reveal MT network, and
GT335 (glutamylation) at 0.1 mg/ml (A lower panel), 0.5 mg/ml (A upper panel and B lower
panel), or 5 mg/ml (B upper panel) to reveal MT glutamylation. TTLL4-transfected cells are
strongly stained with GT335 even at very low concentration (A lower panel), while
nontransfected cells show no detectable MT staining. In contrast, higher concentrations of
GT335 reveal a specific, but faint staining of the MT network in nontransfected cells (B upper
panel). This staining is related to low levels of glutamylation, as it can be diminished by the
deglutamylase CCP5 (B, green cells). These slight differences are barely visible at “standard”
concentrations of GT335 (0.5 mg/ml; B lower panel). Strikingly, the presence of a strong
antigen (A) depletes the antibody from the solution, as nontransfected (white contours) cells
are weakly labeled in B, but not in A (though the same exposure conditions have been
employed). (C) Embryonic rat cortical neurons were fixed after 3 days in vitro using the
DSP-PFA protocol and stained with C105 and GT335 at 0.1 mg/ml. Scale bar in all panels:
10 mm.
