For Research Use Only.
Not for use in diagnostic procedures.

D097-3
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MONOCLONAL ANTIBODY

Anti- GFAP
Code No.
D097-3

Clone
MO389

Subclass
Mouse IgG1

BACKGROUND:Intermediate filaments (IFs)constitute major components of the cytoskeleton and thenuclear envelope in most cell types. Unlike othercytoskeletons such as microtubules and actin filaments, theprotein components of IFs vary in a cell-, tissue-, anddifferentiation-dependent manner. Although IFs were
thought to be relatively stable compared with actin
filaments and microtubules, intensive in vitro
investigations revealed that site-specific phosphorylation
by several kinases, such as protein kinase A (PKA), protein
kinase C (PKC), Ca2+/Calmodulin kinase II (CaMKII),
cdc2 kinase, and Rho-kinase alters dynamically theirstructure and induces filament disruption. Glial fibrillary
acidic protein (GFAP) belongs to IF proteins. GFAP is a
useful marker of mature astrocytes and mutation in the
GFAP gene has recently been associated with Alexander
disease. Inagaki et al. reported that GFAP was
phosphorylated specifically at Thr-7, Ser-13, and Ser-34
by Rho-kinase and Aurora-B and all of these sites were
phosphorylated at the cleavage furrow during cytokinesis.

Quantity
100 L

Concentration
1 mg/mL

SPECIES CROSS REACTIVITY:
Species

Human

Cell

U251

Reactivity on WB

+

Mouse

Rat

-

-

INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

REFERENCES:
1) Kawajiri, A., et al., Mol. Biol. Cell 14, 1489-1500 (2003)
2) Nagata, K., et al., Genes Cells 6, 653-664 (2001)
3) Yasui, Y., et al., J. Cell Biol. 143, 1249-1258 (1998)
4) Kosako, H., et al., J. Biol. Chem., 272, 10333-10336 (1997)
5) Sekimata, M., et al., J. Cell Biol. 132, 635-641 (1996)
6) Matsuoka, Y., et al., EMBO J. 11, 2895-2902 (1992)
Clone MO389 is used in reference number 1) - 5).

SOURCE: This antibody was purified from mouse
ascites fluid using protein A agarose. This hybridoma wasestablished by fusion of mouse myeloma cell SP2/0-Ag14
with Balb/c mouse splenocyte immunized with the KLH
conjugated synthetic peptide CQIRETpSLDTKS.

FORMULATION: 100 g IgG in 100 L volume of
PBS containing 50% glycerol, pH 7.2. No preservative iscontained.

kDa
66
45
30

STORAGE: This antibody solution is stable for one year
from the date of purchase when stored at -20°C.

REACTIVITY: This antibody reacts with non
phosphorylated GFAP and phosphorylated GFAP (~50kDa) on Western blotting and Immunocytochemistry.

Western blot analysis of GFAP
expression in U251 using D097-3.
PROTOCOLS:
SDS-PAGE & Western Blotting

APPLICATIONS:

Western blotting; 0.1 g/mL for chemiluminescence
detection system
Immunoprecipitation; Not tested
Immunohistochemistry; Not tested
Immunocytochemistry; 1 g/mL
Flow cytometry; Not tested
Detailed

procedure
PROTOCOLS.

isprovidedinthefollowing1) Wash the cells 3 times with PBS and suspend with 10
volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2,
250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10%
glycerol) containing appropriate protease inhibitors.
Incubate it at 4oC with rotating for 30 minutes, thensonicate briefly (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4oC
and transfer the supernatant to another tube. Measure theprotein concentration of the supernatant and add the cold
Lysis buffer to make 8 mg/mL solution.

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D097-3
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3) Mix the sample with equal volume of Laemmli’s sample
buffer.
4) Boil the samples for 3 minutes and centrifuge. Load 10
L of the sample per lane in a 1 mm thick
SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF)
membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer
system (Transfer Buffer: 25 mM Tris, 190 mM glycine,
20% MeOH). See the manufacture's manual for precisetransfer procedure.
6) To reduce nonspecific binding, soak the membrane in
10% skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature, or overnight at 4oC.
7) Incubate the membrane with primary antibody diluted
with PBS, pH 7.2 containing 1% skimmed milk assuggested in the APPLICATIONS for 1 hour at room
temperature. (The concentration of antibody will dependon condition.)
8) Wash the membrane with PBS-T [0.05% Tween-20 in
PBS] (5 minutes x 3 times).
9) Incubatethemembranewith
the
1:10,000
HRP-conjugated anti-mouse IgG (MBL; code no. 330)
diluted with 1% skimmed milk (in PBS, pH 7.2) for 1
hour at room temperature.
10) Wash the membrane with PBS-T (10 minutes x 3 times).
11) Wipe excess buffer on the membrane, then incubate itwith appropriate chemiluminescence reagent for 1
minute.
12) Remove extra reagent from the membrane by dabbing
with paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 3 minutes.
14) Develop the film as usual. The condition for exposureand development may vary.
(Positive control for Western blotting; U251)

4) Add the primary antibody diluted with PBS as suggested
in the APPLICATIONS onto the cells and incubate for 1
hour at 4oC (Optimization of antibody concentration or
incubation condition are recommended if necessary.)5) Prepare a wash container such as a 500 mL beaker with astirrer. Then wash the cultured cells on the glass slide bysoaking the slide with a plenty of PBS in the wash
container for 5 minutes. Take care not to touch the cells.
Repeat another washes once more.
6) Add 100 L of 1:100 FITC conjugated anti-mouse IgG(MBL; code no. IM-0819) onto the cells. Incubate for 1
hour at room temperature. Keep out light by aluminum
foil.
7) Wash the slide in a plenty of PBS as in the step 5).
8) Add 1 g/mL of Propidium Iodide (PI) onto the cells.
Incubate for 15 minutes at room temperature.
9) Wash the slide in a plenty of PBS as in the step 5).
10) Wipe excess liquid from slide but take care not to touch
the cells. Never leave the cells to dry.
11) Promptly add mounting medium onto the slide, then put
a cover slip on it.
(Positive control for Immunocytochemistry; U251)

RELATED PRODUCTS:
D097-3 anti-GFAP (MO389)
D098-3 anti-phosphorylated GFAP (Thr7) (TMG7)
MY-01-3 anti-phosphorylated GFAP (Ser8) (YC10)
D121-3 anti-phosphorylated GFAP (The13) (KT13)
D076-3 anti-phosphorylated Vimentin (Ser55) (4A4)
D093-3 anti-phosphorylated Vimentin (Ser71) (TM71)
D094-3 anti-phosphorylated Vimentin (Ser38) (TM38)
D095-3 anti-phosphorylated Vimentin (Ser82) (MO82)
D096-3 anti-phosphorylated Vimentin (Ser6) (MO6)D099-3 anti-phosphorylated Vimentin (Ser33) (YT33)
D122-3 anti-phosphorylated Vimentin (Ser50) (TM50)
PD005
anti-Vimentin Fragment (V1) (polyclonal)

Immunocytochemical detection of GFAP
on 4% PFA fixed U251 using D097-3.
Immunocytochemistry1) Culture the cells in the appropriate condition on a glass
slide. (for example, spread 1x104 of U251 cells for one
slide, then incubate in a CO2 incubator for one night.)
2) Fix the cells by immersing the slide in PBS containing
4% paraformaldehyde for 10 minutes on ice.
3) Immerse the slide in PBS containing 0.1% Triton X-100
for 10 minutes at room temperature.

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