Biology Reference
In-Depth Information
modification sites are detected, especially because of the high-sequence variability
of tubulin within the C-terminal tails, where the modification takes place.
AXO49 is a mouse monoclonal antibody raised against Paramecium axonemes that
recognize
g
-linked glycine side chains of at least three residues (
Callen et al., 1994
). It
has beenmapped to detect side chains of three andmore glycine residues attached to the
Glu 437 of the paramecium
b
-tubulin C-terminal peptide (
Br´ et al., 1998
).
The rabbit polyclonal polyG antibody has been raised against the
-CGGGGGGGGG(-COOH) peptide and detects, similar to the polyE antibody,
C-terminal polyglycine chains (
Rogowski et al., 2009; Wloga et al., 2009
).
None of the antibodies against glycylation are commercially available.
16.2.2
Cell culture and fixation
16.2.2.1
Cell culture and transfection
HeLa cells (ATCC
®
#CCL-2
™
) were grown on glass coverslips (Marienfeld #01 115
20) in DMEM (Life technologies #41965-039) supplemented with 10% fetal bovine
serum (Sigma #F7524) and 2 mM
L
-glutamine (Life technologies #25030-024) at
37
C and in 5% CO
2
. Before use, coverslips were incubated in 60-
m
l drops of a
5
m
g/ml aqueous fibronectin (Sigma #F1141) solution for 1 h at 37
C in a humid
chamber. Coverslips were then transferred to a 24-well plate and washed gently three
times with phosphate-buffered saline (PBS) and left in PBS until the seeding of cells.
The U2OS cell line (ATCC
®
#40342
™
) was grown as described above. Cells
were transfected with expression vectors encoding the murine TTLL4 (
van Dijk
et al., 2007
) or CCP5 (
Rogowski et al., 2010
) genes using jetPei
®
(Polyplus
#101-10) according to manufacturers'
instructions. Cells were fixed 20 h
posttransfection.
Rat cortical neurons were cultured as previously described (
Saudou, Finkbeiner,
Devys, & Greenberg, 1998
) and let to grow until 3 days
in vitro
in neurobasal-B27
medium.
16.2.2.2
Cold methanol fixation
Coverslips were washed once with PBS, then rapidly immersed in
20
C methanol,
and incubated at
20
C for 10 min. Methanol was removed, and the coverslips were
washed three times with PBS and stored in PBS at 4
C if required.
16.2.2.3
PFA fixation
Coverslips were rinsed once with PBS and incubated in 3.7% PFA, 2% sucrose so-
lution for 15 min at room temperature (RT). Coverslips were rinsed three times with
PBS and could be stored in PBS at 4
C if required.
16.2.2.4
DSP-PFA fixation
This method has been described previously (
Bell & Safiejko-Mroczka, 1995
) and
involves several incubation steps that are all performed at RT:
- 10 min incubation in 1 mM DSP in Hank's balanced salt solution (HBSS)
- 10 min incubation in 1 mM DSP in microtubule stabilizing buffer (MTSB)
