Biology Reference
In-Depth Information
to bis-acrylamide ratio, the pH of the Tris buffer, and the type of SDS (see “buffer
composition,”
Section 3.2.1
).
Brains of either 2 days old or adult mice were rapidly homogenized in Laemmli
sample buffer and using an Ultra-Turrax
®
homogenizer (IKA
®
) and heated to 95
C
for 5 min. Subsequently, two short pulses of sonication were applied to fragment ge-
nomic DNA.
The samples were loaded onto the SDS-PAGE gels (for tubulin, 10% acryl amide
gels are used) and run until the bromophenol blue front reaches the bottom of the gel.
The gels were then shortly incubated in transfer buffer, and proteins were transferred
onto a nitrocellulose membrane (Protran BA 85, Whatman, GE Healthcare #10 401
196) for 1 h at 4
C at 100 V and approximately 400 mA using a tank blot system
(Bio-Rad #170-4070).
After transfer, the nitrocellulose membranes were incubated for 1 min in Ponceau
S staining solution and destained twice for 2 min in 1% acidic acid. The membranes
were scanned and transferred to Tris-buffered saline (TBS) containing 0.1% Tween
20 (TBS-T). Subsequently, the membrane was incubated in 5% fat-free dry milk in
TBS-T at RT for 1 h. The primary antibodies were diluted in 2.5% milk-TBS and
were incubated with the membrane at RT for 1 h. After three washes of 5 min with
TBS-T, the membranes were transferred into a dilution of secondary, horseradish
peroxidase (HRP)-labeled antibody (anti-mouse- or anti-rabbit-HRP-conjugated
IgG; GE Healthcare #NA931V or #NA934V, respectively) in TBS-T and incubated
at RT for 45 min. After five washes of 3 min with TBS-T, membranes were devel-
oped by incubation within the ECL Western Blotting Detection Reagents (GE
Healthcare #RPN2209) for 1 min. The membrane was then exposed to photographic
films (Amersham Hyperfilm
™
MP, GE Healthcare #28906844) for 1 min. The films
were developed using a photographic developing machine (Curix60, Agfa) and
scanned with trough light using a linear gray scale gradient.
16.3
BUFFER COMPOSITION
16.3.1
Cell fixation
16.3.1.1
PFA method
PFA-sucrose in PBS
Prepare 4% PFA (Sigma #P6148) in PBS (first dissolve in water by adding
NaOH, bring to pH 7.0, and adjust the concentration with 10
PBS) and then
use this solution to prepare 3.7% PFA, 2% sucrose solution in PBS (can be
aliquoted and stored at
20
C).
16.3.1.2
DSP-PFA method
DSP stock solution
DSP 20 mg/ml in DMSO (50 mM; Perbio Pierce #22585) can be aliquoted
and stored at
20
C.
