I.

Checking if the antibodies using for IHC is suited also for WB:
-Yes, Anti-Stathmin Antibody is suited also for WB but we have just to change the dilution.

II. Instruments those are necessary for the electrophoresis:
Gel electrophoresis is a commonly used technique for the separation of DNA, RNA or protein molecules
using an electric current applied to a gel matrix.
-UV Transilluminator (1):
A transilluminator is commonly used to visualise fluorescent markers used in gel electrophoresis of
nucleic acids and proteins, by emitting a high levels of ultra-violet radiation. For personal safety its
required to use laboratory coats, long sleeved clothes to protect the skin, and glasses and face visor for
the eyes.
- Electrophoresis Power Supplies (2):
An electronic device that supplies electric energy, capable of providing necessary power to perform gel
electrophoresis.
-Protein Gel Electrophoresis Chamber Systems (3):
The chambers are in various sizes and forms (vertical, Horizontal). They can run from 6 samples to 48
samples at times.

Anti-Stathmin Antibody
(Catalog # AB2967, Millipore)
Immunohistochemistry 1:1,000-2,000 dilution
Western Blotting 1:8,000 dilution

.
III. Western Blot Protocol for Figure 2B:
Steps Composition Note
-Place the cell culture dish in ice
-Wash the cells with ice-cold PBS.
-Drain the PBS.
-Add ice-cold lysis buffer.
-Scrape adherent cells off the dish using
a cold plastic cell scraper.
-Transfer gently the cell suspension into
a pre-cooled microfuge tube. And
Maintain constant agitation for 30
minutes at 4C.
-Centrifuge in a microcentrifuge at 4C
for 15 minutes at 14,000 rpm.
lysis buffer:
- Nonidet-P40 (NP-40) buffer:
150 mM sodium chloride
1.0% NP-40 (Triton X-100
can be substituted for NP-
40)
50 mM Tris, pH 8.0
-Tris-HCl buffer :
20 mM Tris-HCl, pH 7.5
-100 mM NaCl
-5 mM MgCl2
-0.5% NaDOC (sodium
DeOxyCholate)
-1 mM phenylmethylsulfonyl
fluoride (PMSF).
-2 mM orthovanadate.
Preparation of
lysate from cell
culture ( GN-11
cell)
-Dissect the tissue with clean tool on ice,
and as quickly as possible to prevent
degradation by proteases.
-Place the tissue in round bottom
microfuge tubes of Eppendorf tubes.
-Immerse in liquid nitrogen to snap
freeze.
-For 5 mg piece of tissue, add 300 l
lysis buffer rapidly to the tube.
- homogenize with an electric
homogenizer.
- Rinse the blade twice with another
2x300 l lysis buffer.
-Then maintain constant agitation for 2
hours at 4C.
-Centrifuge for 15 minutes at 14,000
rpm at 4C in a microcentrifuge.
-Remove the tubes from the centrifuge
and place on ice.
-lysis buffer: Idem to the
precedent.
- Electric homogenizer.
- Microcentrifuge

Preparation for
Olfactory bulb
(OB) tissue.
- Use BCA (bicinchoninic acid)
Protein Assay Kit (Catalog # BCA-1,
Sigma-Aldrich) for protein
determination, by following the product
Instructions
BCA Protein Assay Kit
(Catalog # BCA-1, Sigma-Aldrich)
Determination of
protein
concentration
- Proteins are boiled in Laemmli buffer Laemmli buffer: Preparation of
at 95-100C for 5 minutes.
-then Proteins are subjected to 15%
(stathmin) SDS-PAGE.

2% sodium dodecyl
sulfate,
50 mM Tris-HCl (pH
7.4),
20% -mercaptoethanol,
20% glycerol

samples for
loading into gels
-A standard migration buffer (also called
running bugger) for PAGE is 1X Tris-
glycine.
- Check the pH; it should be around 8.3.
- Run the gel for the recommended time
as instructed by the manufacturer.
- When the dye molecule (molecular
weight markers) reached the bottom of
the gel, the power is turned off. Proteins
will slowly elute from the gel at this
point, so do not store the gel; proceed
immediately to transfer.

Tris-glycine:
25 mM Tris base
190 mM glycine
0.1% SDS




Preparation of
PAGE gels
- Block with 5% dry milk (block
nonspecific binding sites).

Blocking the
membrane
-Cut the membrane (PVDF) to the
appropriate size.
- Soak it in methanol for 1-2 minutes.
-Incubate in ice cold transfer buffer for
5 minutes.
-PVDF (positively-charged
nylon) membrane.
- Transfer buffer ( standard recipe):
48 mM Tris.
39 mM glycine.
0.04% SDS, 20%.
Methanol.
Membrane
preparation
-To check for success of transfer, wash
the membrane in TBST.
-Dilute the stock Ponceau Red 1:10.
(The stock is made of 2% Ponceau S in
30% trichloracetic acid and 30%
sulfosalicylic acid).
- Incubate on an agitator for 5 minutes.
- Wash extensively in water until the
water is clear and the protein bands are
well defined.
- The membrane may be destained
completely by repeated washing in
TBST or water. When using a PVDF
membrane, re-activate the membrane
with methanol then wash again in
TBST.
- TBST buffer:
20 mM Tris,
150 mm NaCl,
0.1% Tween 20 (pH 7.4)

Visualization of
proteins in
membranes:
Ponceau Red


- Dilute Anti-Stathmin Antibody
(Catalog # AB2967, Millipore) in TBST at
1:1000 at 4C.

Anti-Stathmin Antibody
(Catalog # AB2967, Millipore)
Incubation with
the primary
antibody
- Wash the membrane several times in
TBST while agitating, 5 minutes or
more per wash, to remove residual
primary antibody.
- Dilute the Goat Anti-Rabbit IgG
(H+L) HRP conjugate Secondary
Antibody (Catalog # AP307P , Millipore)
in TBST at 1:5,000 for 1-2 hours, room
temperature, with agitation.
- Wash several times in TBST to remove
residual secondary antibody.
Goat Anti-Rabbit IgG (H+L)
HRP conjugate Secondary
Antibody (Catalog # AP307P,
Millipore)
Incubation with
secondary
antibody
- Use ECL detection system following
the manufacturer guide (Amersham
Biosciences).
Detection kits