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

PM056
Page 1 of 3

POLYCLONAL ANTIBODY

Anti-Sox2 pAb
Code No.
PM056

Quantity100 µL

BACKGROUND: Sox2, a member of the SRY-related
HMG-box (SOX) family of transcription factors, isexpressed in embryonic inner cell mass, epiblast,extraembryonic mesoderm. It is one of the four factors(Oct3/4, Sox2, Klf4, c-Myc) that have been used to form
iPS cells (induced pluripotent stem cells) from fibroblasts.
Sox2 and Oct3/4 works together to control a transcriptionalregulatory network that regulates the expression of otheressential genes, and plays important roles in early
embryonic development.

SOURCE: This antibody was purified from rabbit serum
using affinity column. The rabbit was immunized withpartial recombinant mouse Sox2 protein.

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

Form
Affinity Purified

SPECIES CROSS REACTIVITY:
Species

HumanCells

NCCIT

Reactivity
on WB+Mouse

Rat Chicken**

293T,
P19*,
NIH/3T3, Not
HeLa transfectant WR19L tested-+-*This antibody shows weak reactivity to differentiated P19.
**The reactivity to chicken is reported in reference 2).

REFERENCES:
1) Suzuki, Y., et al., Int. J. Oncol. 42, 161-167 (2013) [WB]
2) Gao, Y., et al., Eur. J. Histochem. 56, e33 (2012) [IC]3) Maekawa, M., et al., Nature 474, 225-229 (2011)4) Niwa, H., Development 134, 635-646 (2007)
5) Yu, J., et al., Science 318, 1917-1920 (2007)
6) Masui, S., et al., Nat. Cell Biol. 9, 625-635 (2007)
7) Avilion, A. A., et al., Genes & Dev. 17, 126-140 (2003)

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

This antibody is used in the reference 1)-3).

REACTIVITY: This antibody reacts with mouse Sox2

RELATED PRODUCTS:(35 kDa) on Western blotting, Immunohistochemistry,
Immunocytochemistry and Immunoprecipitation. The
reactivity to human Sox2 was confirmed by Westernblotting and Immunoprecipitation.

M164-3
PM048
PM055

Anti-Oct3/4 mAb (2F12)Anti-Oct3/4 pAb (polyclonal)
Anti-Lin28 pAb (polyclonal)

APPLICATIONS:
Western blotting; 1:1,000 for a chemiluminescence
detection system
Immunoprecipitation; 2 µL/300 µL of cell extract from3 x 106 cells
Immunohistochemistry; 1:500
Heat treatment is necessary.
Microwave oven; 500W
Frozen section: 1 mM EDTA (pH 8.0), 3 minutes
Paraffin section: 10 mM citrate buffer (pH 6.3), 20 minutes
Immunocytochemistry; 1:200
Flow cytometry; Not tested
Detailed procedures are provided in the following

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

Immunohistochemical detection ofSox2 on frozen section of mouseembryo (E13) with PM056.
Fluorescence Microscope: Axiovert200
Filter set:Carl Zeiss Filter sets No.26
Lens: Plan-NEOFLUAR (Carl Zeiss), x20, NA=0.5

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

PM056
Page 2 of 3

PROTOCOLS:
Immunohistochemical staining for frozen sections
For 4% paraformaldehyde fixed section
1) Wash the slide in PBS for 15 minutes.
2) Heat treatment
Heat treatment by Microwave:
Place the slides put on staining basket in 1 L beaker with500 mL of 1 mM EDTA (pH 8.0). Cover the beakerwith plastic wrap, then process the slides 3 minutes at500 W with microwave oven. Let the slides cool downin the beaker at room temperature for about 40 minutes.
3) Immerse the slide in PBS containing 0.1% Tween-20(PBS-T) for 30 minutes at room temperature.
4) Remove the slides from PBS-T, wipe gently around eachsection and cover tissues with blocking buffer (PBScontaining 2% FCS, 0.1% Tween-20) for 5 minutes toblock non-specific staining. Do not wash.
5) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with blocking buffer
as suggested
in theAPPLICATIONS.
6) Incubate the sections for 1 hour at room temperature.
7) Wash the slides 2 times in PBS-T for 5 minutes each.
8) Wipe gently around each section and cover tissues with
1:500 Alexa Fluor® 647 conjugated anti-rabbit IgG(Invitrogen; code no. A21245). Incubate for 30 minutes atroom temperature. Wash as in step 7).
9) Wipe excess liquid off the slide but take care not to touch
the section. Never leave the section to dry.
10) Promptly add mounting medium onto the slide, then put acover slip on it.
(Positive control for Immunohistochemistry; Mouse embryo)Heat treatment by Microwave:
Place the slides put on staining basket in 1 L beakerwith 500 mL of 10 mM citrate buffer (pH 6.3). Coverthe beaker with plastic wrap, then process the slides for
20 minutes at 500 W with microwave oven. Let theslides cool down in the beaker at room temperature forabout 40 minutes.
5) Remove the slides from the citrate buffer (pH 6.3) andcover each section with 3% H2O2 for 10 minutes at room
temperature to block endogenous peroxidase activity.
Wash 2 times in PBS for 5 minutes each.
6) Remove the slides from PBS, wipe gently around eachsection and cover tissues with blocking buffer (20 mM
HEPES, 1% BSA, 135 mM NaCl) for 5 minutes to block
non-specific staining. Do not wash.
7) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with blocking buffer
as suggested
in theAPPLICATIONS.
8) Incubate the sections for 1 hour at room temperature.
9) Wash the slides 3 times in PBS for 5 minutes each.
10) Wipe gently around each section and cover tissues withHistostar (Ms + Rb) (MBL; code no. 8460). Incubate for 1
hour at room temperature. Wash as in step 9).
11) Visualize by reacting for 10 minutes with DAB substratesolution (MBL; code no. 8469). *DAB is a suspect carcinogenand must be handled with care. Always wear gloves.

12) Wash the slides in water for 5 minutes.
13) Counter stain in hematoxylin for 1 minute, wash the slides
3 times in water for 5 minutes each, and then immerse theslides in PBS for 5 minutes.
14) Dehydrate by immersing in Ethanol 3 times for 3 minutes
each, followed by immersing in Xylene 3 times for 3
minutes each. Now ready for mounting.
(Positive control for Immunohistochemistry; mouse embryo)

Immunohistochemical detection ofSox2 on paraffin embedded sectionof mouse embryo (E13) with PM056.
Immunohistochemical staining for paraffin-embedded
sections
1) Deparaffinize the sections with Xylene 3 times for 3-5
minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minuteseach.
3) Wash the slides 3 times in PBS for 3-5 minutes each.
4) Heat treatment

Immunocytochemical
detection
Sox2 in P19 with PM056.

ofImmunocytochemistry1) 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.

PM056
Page 3 of 35) Immerse the slide in PBS containing 0.2% Triton X-100for 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 µL of 1:500 Alexa Fluor® 488 conjugatedanti-rabbit IgG (Invitrogen; code no. A110374) dilutedwith 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)

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 10 µLof 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 transfersystem (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 room
temperature, or overnight at 4°C.
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 Anti-IgG (Rabbit)
pAb-HRP (MBL; code no. 458) diluted with 1% skimmed
milk (in PBS, pH 7.2) for 30 minutes 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 3 minutes. Develop the film under usual settings. The
conditions for exposure and development may vary.

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)
containingproteaseinhibitors
atappropriate
concentrations. Incubate it at 4°C with rotating for 30minutes; thereafter, briefly sonicate the mixture (up to 10seconds).
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 200 µL of the supernatant. Mix
well and incubate with gentle agitation for 60-120 minutesat 4°C.
4) Add 20 µL 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) Wash the beads 3-5 times with the cold Lysis buffer(centrifuge the tube at 2,500 x g for 10 seconds).
6) Resuspend the beads in 20 µL of Laemmli’s sample buffer,boil for 3-5 minutes, and centrifuge for 5 minutes. Use 20µL/lane for the SDS-PAGE analysis.
(See SDS-PAGE & Western blotting.)(Positive control for Immunoprecipitation: HeLa)(Positive controls for Western blotting; NCCIT and HeLa)

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