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

D130-3
Lot 036~
Page 1

MONOCLONAL ANTIBODY

Anti-Runx2 (Cbfa1) mAb
Code No.
D130-3Clone
8G5

Subclass
Mouse IgG2b 

BACKGROUND: Runx2, also known as Cbfa1 orPEBP2A, is an essential transcription factor of skeletal
tissues that is involved in the regulation of osteoblast
differentiation and bone formation. Runx2-null mice haveneither bone tissue nor osteoblasts. FGF receptor signaling,
TGF-, and BMP all activate transcription of Runx2,
resulting in inhibition of myogenesis and myogenic
differentiation. BMP-induced Runx2 cooperates withBMP-activated Smads to induce osteogenesis. Runx2 has
also been shown to play a role in the regulation ofchondrocyte hypertrophy and tooth eruption.

SOURCE: This antibody was purified from hybridoma(clone 8G5) supernatant using protein A agarose. Thishybridoma was established by fusion of mouse myeloma
cell X63-Ag8.653 with Balb/c mouse splenocyte
immunized with the recombinant Runx2.

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

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

REACTIVITY: This antibody reacts with human
Runx2/Cbfa1 (66 kDa) and mouse Runx2/Cbfa1 (55 kDa)
on Western blotting.

Quantity
100 L

Concentration
1 mg/mL

SPECIES CROSS REACTIVITY:
Species

Human

Mouse*

Rat**Cells

Saos2

2T3, C2C12

Not tested

Reactivity on WB++*This monoclonal antibody is widely used to detectmouse Runx2/Cbfa15), 7)-9), 11).
**The cross reactivity to rat Runx2/Cbfa1 is reported1), 3), 6).

REFERENCES:
1) Beier, E. E., et al., Environ. Health Perspect. 121, 97-104 (2013) [IHC]
2) Underwood, K. F., et al., J. Bone Miner. Res. 27, 913-925 (2012)[WB, ELISA]
3) Hosoya, A., et al., J. Histochem. Cytochem. 60, 861-73 (2012) [IHC]
4) Ali, S. A., et al., J. Cell Sci. 125, 2732-2739 (2012) [IC]5) Fulzele, K., et al., Cell 142, 309-319 (2011) [ChIP]
6) Hirata, A., et al., J. Histochem. Cytochem. 57, 397-403 (2009) [IHC]
7) Tominaga, H., et al., Mol. Biol. Cell 19, 5373-5386 (2008) [WB]
8) Ohba, S., et al., FASEB J. 21, 1777-1787 (2007) [WB, IP, ChIP]
9) Wang, Q., et al., J. Biol. Chem. 282, 10742-10748 (2007) [ChIP]
10) Lau, Q. C., et al., Cancer Res. 66, 6512-6520 (2006) [WB, IC]11) Kaneki, H., et al., J. Biol. Chem. 281, 4326-4333 (2006) [WB]
12) Wee, H. J., et al., EMBO Rep. 3, 967-974 (2002) [WB, IP]
13) Zhang, Y. W., et al., PNAS 97, 10549-10554 (2000) [WB]
As this monoclonal antibody is widely used, many
researches have been reported. These references are a partof such reports.

APPLICATION-CONFIRMED:
Western blotting; 1 g/mL
Detailed

procedure
PROTOCOL.

isprovidedinthefollowing

kDa
212
170
116

APPLICATIONS-REPORTED:

76

Immunoprecipitation; Reference 8) and 12)
Immunohistochemistry; Reference 1), 3) and 6)
Immunocytochemistry; Reference 4) and 10)
ELISA; Reference 2)
Chromatin Immunoprecipitation; Reference 5), 8) and 9)

53

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

Western blot analysis of Runx2
expression in Saos2 cells using
D130-3.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL https://ruo.mbl.co.jp

e-mail support@mbl.co.jp

D130-3
Lot 036~
Page 2The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

PROTOCOL:
SDS-PAGE & Western Blotting1) 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 at4°C with rotating for 30 minutes, then sonicate briefly (upto 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°Cand transfer the supernatant to another tube. Measure theprotein concentration of the supernatant and add the Lysis
buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s sample
buffer.
4) Boil the samples for 2 minutes and centrifuge. Load 10 Lof 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 transfersystem (Transfer Buffer: 25 mM Tris, 190 mM glycine,
20% methanol). 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 4°C.
7) Incubate the membrane with primary antibody diluted
with 1% skimmed milk (in PBS, pH 7.2) as suggested inthe APPLICATIONS for 1 hour at room temperature.
(The concentration of antibody will depend on theconditions.)
8) Wash the membrane with PBS-T [0.05% Tween-20 inPBS] (5 minutes x 6).
9) Incubate the membrane with the 1:10,000 Anti-IgG(Mouse) pAb-HRP (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 (5 minutes x 6).
11) Wipe excess buffer on the membrane, then incubate it withappropriate chemiluminescence reagent for 1 minute.
Remove extra reagent from the membrane by dabbingwith paper towel, and seal it in plastic wrap.
12) Expose to an X-ray film in a dark room for 5 minutes.
Develop the film as usual. The condition for exposure anddevelopment may vary.
(Positive control for Western blotting; Saos2)

RELATED PRODUCTS:
Please visit our web site https://ruo.mbl.co.jp/.

220705-6