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

PM057
Lot 003~
Page 1POLYCLONAL ANTIBODY

Anti-KLF4 pAb
Code No.
PM057

Quantity
100 LForm
Affinity Purified

BACKGROUND: KLF4 is a zinc-finger transcription

REFERENCES:

factor expressed in the epithelium of the skin, lungs,
intestine and several other organs. KLF4-KO mouse diesafter birth for the abnormal formation of the skin. It is
known that Klf4 cooperates with Oct3/4 and Sox2, and it is
one of the factors that have been used to form iPS cells(induced pluripotent stem cells) from fibroblasts.

1) Takahashi, K., et al., Cell 131, 861-872 (2007)
2) Nakatake, Y., et al., Mol. Cell Biol. 26, 7772-7782 (2006)
3) Katz, J. P., et al., Development 129, 2619-2628 (2002)

SOURCE: This antibody was purified from rabbit serum
using affinity column. The rabbit was immunized with therecombinant full length of human KLF4.

RELATED PRODUCTS:
Please visit our web site https://ruo.mbl.co.jp/.
The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

FORMULATION: 100 L volume of PBS containing

kDa

50% glycerol, pH 7.2. Contains no preservative.

75

1234

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

50

REACTIVITY: This antibody reacts with human and37

mouse KLF4 on Western blotting.

APPLICATIONS:
Western blotting; 1:1,000
Immunoprecipitation; 2 L/300 L of cell extract from3 x 106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1:200
Flow cytometry; Not tested

Westernblot
analysisofKLF4
expression on human KLF4 transfectant(1), mouse KLF4 transfectant (2), NCCIT(3) and HeLa (4) using PM057.

PROTOCOLS:
SDS-PAGE & Western Blotting

Detailed procedures are provided in the following
PROTOCOLS.

SPECIES CROSS REACTIVITY:
SpeciesCells

HumanMouse

NCCIT, HeLa,transfectant
transfectant

Reactivity on WB+Rat
Not tested+

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

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 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 4oC.
5) Incubate the membrane for 1 hour at room temperaturewith 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).

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

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PM057
Lot 003~Page 27) 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 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 controls for Western blotting; transfectant,NCCIT, HeLa)

kDa

1

275
KLF450IgG heavy chain

37
Immunoprecipitation of KLF4 from HeLa
with Normal rabbit IgG (1) or PM057 (2).
After immunoprecipitated with the antibody,immunocomplex was resolved on SDS-PAGE
and immunoblotted with PM057.
Immunoprecipitation
1) Wash cells (approximately 1 x 107 cells) 3 times with PBS
and 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 30minutes; thereafter, briefly sonicate the mixture (up to 10seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 oCand 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.)(Positive control for Immunoprecipitation; HeLa)

Immunocytochemicaldetection
KLF4 in HeLa with PM057.

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.
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) 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; HeLa)

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