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

PM048
Page 1 of 3

POLYCLONAL ANTIBODY

Anti-Oct3/4 pAb
Code No.
PM048

Quantity100 L

BACKGROUND: The POU family transcription factor
Oct3/4, termed as Oct3 or Oct4, encoded by Pou5f1, isexpressed in totipotent/pluripotent early embryonic cells. Itis also expressed in embryonic stem (ES) cells andembryonal carcinoma (EC) cells, but its expression
diminishes when these cells differentiate and lose
pluripotency. Oct3/4 contains three functionallycharacterized domains, the transcriptional activation
domain of N- and C-terminal region and the POU
DNA-binding domain. The POU domain binds to an
octamer sequence, ATTTGCAT. Several target genes ofOct3/4, such as Sox2, contains an octamer element capableof binding Oct3/4. These sites are important fortranscriptional activity. Induced pluripotent stem (iPS) cells
can be generated from mouse embryonic or adultfibroblasts by induction of four factors, Oct3/4, Sox2,
c-Myc, and Klf4. Oct3/4 regulates a expression of Tcl1 (Tcell lymphoma break point) and Nanog and contributes tocell proliferation and stabilization of cell pluripotency.
Two transcription factors Oct3/4 and Sox2 works together
to control a transcriptional regulatory network that
regulates the expression of other essential genes.

SOURCE: This antibody was purified from rabbit serum

Form
Affinity Purified

SPECIES CROSS REACTIVITY:
Species
Cells
Reactivity
on WB

HumanMouse

Rat Hamster

HL-60,MEF,
P19
Transfectant 293T,
NIH/3T3, Rat1
Transfectant
HeLa
WR19L+-+-CHO-INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

REFERENCES:
1) Boer, B., et al., Nucleic Acids Res. 35, 1773-1786 (2007)
2) Takahashi, K., and Yamanaka, S., Cell 126, 663-676 (2006)
3) Chew, J., et al., Mol. Cell Biol. 25, 6031-6046 (2005)
4) Niwa, H., et al., Cell 123, 917-929 (2005)
5) Saijoh, Y., et al., Genes to Cells 1, 239-252 (1996)
6) Shimazaki, T., et al., EMBO. J. 12, 4489-4498 (1993)
7) Okamoto, K., et al., Cell 60, 461-472 (1990)

PROTOCOLS:

using affinity column. The rabbit was immunized withrecombinant N-terminal of mouse Oct3/4 corresponding to1-134 aa.

12

FORMULATION: 100 L volume of PBS containing
50% glycerol, pH 7.2. No preservative is contained.

STORAGE: This antibody solution is stable for one year
from the date of purchase when stored at -20°C.
3

REACTIVITY: This antibody reacts with Oct3/4 on
Westernblotting,Immunoprecipitation,Immunocytochemistry and Immunohistochemistry.

APPLICATIONS:
Western blotting; 1:1,000
Immunoprecipitation; 2 L/300 L of cell extract from3 x 106 cells
Immunohistochemistry; 1:500
Immunocytochemistry; 1:500
Flow cytometry; Not tested
Detailed procedure is provided in the following
PROTOCOLS.

-1: DAPI
2: anti-Oct3/4
3: Merge

Immunocytochemical
detectionofOct3/4 in mouse ES cell with PM048.
This data was kindly provided by Yoshiba and Hamada
M.D., Ph.D. (Developmental Genetics Group, Graduate
School of Frontier Biosciences, Osaka University)

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

e-mail support@mbl.co.jp

PM048Page 2 of 3

Immunocytochemistry
1) Fix the cells by immersing the glass slide in PBS
containing 4% paraformaldehyde (PFA) for 20 minutes atroom temperature.
2) Rinse the glass slide in 100% methanol.
3) Rinse the glass slide in PBS containing 0.1% Triton
X-100.
4) Immerse the slide 5% skimmed milk in PBS containing
0.1% Triton X-100 for 1 hour at room temperature.
5) Add 30 L of the primary antibody diluted with 5%
skimmed milk, 0.1% Triton X-100 in PBS as suggested in
the APPLICATIONS onto the cells and incubate for 30
minutes at room temperature. (Optimization of antibody
concentration or incubation condition is recommended ifnecessary.)6) The glass slide was washed with PBS for 30 minutes atroom temperature.
7) Add 30 L of 1:1,000 Alexa Fluor® 488 conjugatedanti-rabbit IgG (Invitrogen; code no. A11008) diluted
with PBS onto the cells. Incubate for 1 hour at room
temperature. Keep out light by aluminum foil.
8) The glass slide was washed with PBS for 30 minutes atroom temperature.
9) Wipe excess liquid off the slide but take care not to touch
the cells. Never leave the cells to dry.
10) Promptly add mounting medium onto the slide, then put acover slip on it.

2) Permeabilize a section with PBS containing 0.2% Triton
X-100 for 10minutes.
3) Wipe gently around each section and cover tissues withblocking buffer [5% skimmed milk in PBS containing
0.1% Triton X-100] for 30 minutes to block non-specificstaining. Do not wash.
4) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with blocking buffer
as suggested in theAPPLICATIONS.
5) Incubate the sections at room temperature for 1 hour.
6) Wash the slides in PBS-T [PBS containing 0.1% Triton
X-100] (15 minutes x 3 times).
7) Wipe gently around each section and cover tissues with
Alexa Fluor® 488 labeled anti-Rabbit IgG (Invitrogen;
A11008) diluted 1:1,000 with PBS-T. Incubate for 30
minutes at room temperature.
8) Wash the slides in PBS-T (15 minutes x 2 times).
9) Wipe gently around each section and cover tissues withDAPI in PBS-T. Incubate for 5 minutes.
10) Wash the slides in PBS-T for 5 minutes.
11) Mount the slides, then put a cover slip on it.
(Positive control for Immunohistochemistry; mouse embryo)(Positive control for Immunocytochemistry; mouse ES cell)

123

1: DAPI
2: anti-Oct3/4
3: Transmission light

Immunohistochemicaldetection
ofOct3/4 on frozen section of mouseembryo (E3.5) with PM048.
This data was kindly provided by Takaoka Ph.D. andHamada M.D., Ph.D. (Developmental Genetics Group,Graduate School of Frontier Biosciences, Osaka
University)

Immunohistochemical staining for frozen sections
For 4% paraformaldehyde fixed section
1) Wash the slide in PBS (10 minutes x 2 times).

SDS-PAGE & Western blotting1) Wash cells (approximately 1 x 107 cells) 3 times with PBS
and resuspend them in 1 mL of Laemmli’s sample buffer.
2) Boil the samples for 2 minutes and centrifuge. Load 20 Lof the sample per lane on a 1-mm-thick
SDS-polyacrylamide gel and carry out electrophoresis.
3) 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% Methanol). See the manufacturer's manual for precisetransfer procedure.
4) To reduce nonspecific binding, soak the membrane in 5%skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature, or overnight at 4°C.
5) Incubate the membrane for 1 hour at room temperature

PM048Page 3 of 3with primary antibody diluted with 1% skimmed milk (inPBS, pH 7.2) as suggested in the APPLICATIONS. (Theconcentration of antibody will depend on the conditions.)
6) Wash the membrane with PBS-T [0.05% Tween-20 inPBS] (5 minutes x 3 times).
7) Incubate the membrane with 1:10,000 of Anti-IgG(Rabbit) pAb-HRP (MBL; code no. 458) diluted with 1%
skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature.
8) Wash the membrane with PBS-T (5 minutes x 3 times).
9) Wipe excess buffer off the membrane, and incubate
membrane with an appropriate chemiluminescencereagent for 1 minute.
10) Remove extra reagent from the membrane by dabbing
with a paper towel, and seal it in plastic wrap.
11) Expose the membrane onto an X-ray film in a dark roomfor 10 minutes. Develop the film under usual settings. The
conditions for exposure and development may vary.
(Positive control for Western blotting; P19)

kDa

1

27550
Oct3/4
3725
Immunoprecipitation of Oct3/4 from P19
with Normal rabbit IgG (1) or PM048 (2).
After immunoprecipitated with the antibody,immunocomplex was resolved on SDS-PAGE
and immunoblotted with M164-3.

discard the supernatant.
6) Resuspend the beads with the cold Lysis buffer.
7) Centrifuge the tube at 2,000 x g for 10 seconds anddiscard the supernatant.
8) Repeat steps 6)-7) 3-5 times.
9) Resuspend the agarose in 20 L of Laemmli’s sample
buffer, boil for 3-5 minutes, and centrifuge for 5 minutes.
10) Load 10 L of the sample per lane on a 1-mm-thick
SDS-polyacrylamide gel and carry out electrophoresis.
11) 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 manufacturer's manual for precisetransfer procedure.
12) To reduce nonspecific binding, soak the membrane in 5%
skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature, or overnight at 4°C.
13) Incubate the membrane with 1 g/mL of Anti-Oct3/4 mAb(MBL; code no. M164-3) diluted with 1% skimmed milk(in PBS, pH 7.2) for 1 hour at room temperature. (Theconcentration of antibody will depend on the conditions.)
14) Wash the membrane with PBS-T [0.05% Tween-20 inPBS] (5 minutes x 3 times).
15) Incubate the membrane with 1:10,000 of 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.
16) Wash the membrane with PBS-T (5 minutes x 3 times).
17) Wipe excess buffer off the membrane, and incubate
membrane with an appropriate chemiluminescencereagent for 1 minute.
18) Remove extra reagent from the membrane by dabbing
with a paper towel, and seal it in plastic wrap.
19) Expose the membrane onto an X-ray film in a dark roomfor 3 minutes. Develop the film under usual settings. The
conditions for exposure and development may vary.
(Positive control for Immunoprecipitation: P19)

RELATED PRODUCTS:
Please visit our website at http://ruo.mbl.co.jp/.

Immunoprecipitation
1) Wash cells (approximately 1 x 107 cells) 3 times with PBS
and suspend with 10 volumes of cold Lysis buffer [50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% NP-40]
containing appropriate protease inhibitors. Incubate it at
4°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°C
and transfer the supernatant to another fresh tube.
3) Add primary antibody as suggested in theAPPLICATIONS into 300 L of cell extract. Mix welland incubate with gentle agitation for 30-120 minutes at4°C.
4) Add 20 L of 50% protein A agarose beads resuspended
in the cold Lysis buffer. Mix well and incubate with gentle
agitation for 60 minutes at 4°C.
5) Centrifuge the tube at 2,000 x g for 10 seconds and190806-3