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

PM058
Page 1 of 2

POLYCLONAL ANTIBODY

Anti-mouse Nanog
Code No.
PM058

Quantity
100 LForm
Affinity Purified

BACKGROUND: Embryonic stem (ES) cells are

REFERENCES:

pluripotent stem cells derived from the inner cell mass ofthe blastocyst. Nanog containing a homeobox domain is atranscription factor expressed in ES cells, and it is required
for maintenance of pluripotency in mouse epiblast and EScells. Pluripotency of mouse ES cells can be maintained byleukemia inhibitory factor (LIF), but Nanog is capable ofmaintaining ES cell self-renewal independently of LIF.

1) Junying, Yu., et al., Science 318, 1917-1920 (2007)
2) Mitsui, K., et al., Cell 113, 631-642 (2003)
3) Chambers, I., et al., Cell 113, 643-655 (2003)

SOURCE: This antibody was purified from rabbit serumkDa

1

50using affinity column. The rabbit was immunized with therecombinant full length of mouse Nanog.

FORMULATION: 100 L volume of PBS containing

2mouse Nanog
3750% glycerol, pH 7.2. Contains no preservative.

STORAGE: This antibody solution is stable for one year

25ofrom the date of purchase when stored at -20 C.

REACTIVITY: This antibody reacts with mouse Nanog
on
Westernblotting,Immunocytochemistry.

Immunoprecipitation

andWestern blot analysis of mouse Nanog
expression on His tagged mouse Nanogtransfectant (1), and P19 (2) using PM058.

PROTOCOLS:
APPLICATIONS:SDS-PAGE & Western Blotting

Western blotting; 1:500 for a chemiluminescence detection
systemImmunoprecipitation; 2 L/300 L of cell extract from3 x 106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1:200
Flow cytometry; Not tested

1) 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 10 Lof 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% MeOH). See the manufacturer's manual for precisetransfer procedure.
4) To reduce nonspecific binding, soak the membrane in 10%
skimmed milk (in PBS, pH 7.2) for 1 hour at roomtemperature, or overnight at 4oC.
5) Incubate the membrane for 1 hour at room temperature
with primary antibody diluted with PBS (pH 7.2)
containing 1% skimmed milk as suggested in theAPPLICATIONS. (The concentration 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 HRP-conjugatedanti-rabbit IgG (MBL; code no. 458) diluted with 1%
skimmed milk (in PBS, pH 7.2) for 1 hour at roomtemperature.

Detailed procedures are provided in the following

PROTOCOLS.
SPECIES CROSS REACTIVITY:
Species

Human

Mouse

RatCells

Not Tested

P19,transfectant

Not Tested

Reactivity on WB+INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL https://ruo.mbl.co.jp
e-mail support@mbl.co.jp, TEL 052-238-1904

PM058Page 2 of 28) 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 3 minutes. Develop the film under usual settings. The
conditions for exposure and development may vary.
(Positive controls for Western blotting; transfectant, P19)

kDa
75

1

Immunocytochemical detection
mouse Nanog in P19 with PM058.

of250

ImmunocytochemistryIgG heavy chain
mouse Nanog

37
Immunoprecipitation of mouse Nanog
from P19 with Normal rabbit IgG (1) orPM058 (2). After immunoprecipitated with theantibody, immunocomplex was resolved onSDS-PAGE and immunoblotted with PM058.
Immunoprecipitation
1) Wash cells (approximately 1 x 107 cells) 3 times with PBSand resuspend them in 1 mL of cold Lysis buffer (50 mM
Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40)
containing
protease
inhibitors
atappropriate
concentrations. Incubate it at 4oC with rotating for 30
minutes; thereafter, briefly sonicate the mixture (up to 10seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4oCand transfer the supernatant to another tube.
3) Add primary antibody as suggested in theAPPLICATIONS into 300 L of the supernatant. Mix
well and incubate with gentle agitation for 30-120 minutes
at 4oC. Add 20 L of 50% protein A agarose beads
resuspended in the cold Lysis buffer. Mix well andincubate with gentle agitation for 60 minutes at 4oC.
4) Wash the beads 3-5 times with the cold Lysis buffer(centrifuge the tube at 2,500 x g for 10 seconds).
5) Resuspend the beads in 20 L of Laemmli’s sample buffer,boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10
L/lane for the SDS-PAGE analysis.
(See SDS-PAGE & Western blotting.)

1) Culture the cells in the appropriate condition on a glassslide. (for example, spread 1 x 104 cells for one slide, thenincubate in a CO2 incubator for one night.)
2) Wash the glass slide 2 times with PBS.
3) Fix the cells by immersing the slide in PBS containing 4%paraformaldehyde for 10 minutes at room temperature.
4) Wash the glass slide 3 times with PBS.
5) Immerse the slide in PBS containing 0.2% Triton X-100
for 10 minutes at room temperature.
6) Wash the glass slide 2 times with PBS.
7) Add the primary antibody diluted with PBS containing
2% FCS as suggested in the APPLICATIONS onto thecells and incubate for 1 hour at room temperature(Optimization of antibody concentration or incubation
condition are recommended if necessary.)8) Wash the glass slide 2 times with PBS.
9) Add 100 L of 1:500 Alexa Fluor® 488 conjugatedanti-rabbit IgG (Invitrogen; code no. A110374) diluted
with PBS onto the cells. Incubate for 30 minutes at room
temperature. Keep out light by aluminum foil.
10) Wash the glass slide 2 times with PBS.
11) Wipe excess liquid off the slide but take care not to touch
the cells. Never leave the cells to dry.
12) Promptly add mounting medium onto the slide, then put acover slip on it.
(Positive control for Immunocytochemistry; P19)

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