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

PM062
Lot 004~
Page 1POLYCLONAL ANTIBODY

Early Endosome Marker

Anti-EEA1 pAb
Code No.
PM062

Quantity
100 LForm
Affinity Purified

BACKGROUND: The early endosome is a cellular

REFERENCES:

compartment inside eukaryotic cells. It is receiving
endocytosed material and sorting them for vesiculartransport to late endosomes and lysosomes or for recycling
to the plasma membrane. EEA1 (Early Endosome Antigen1) is a 170 kDa coiled-coil protein, which is required forvesicular transport of proteins through early endosomes. It
binds membrane lipids through its FYVE domain.

1) Abe, S., et al., J. Biol. Chem. 292, 4089-4098 (2017) [IC]2) Gaullier, J. M., et al., J. Biol. Chem. 275, 24595-24600 (2000)
3) Mu, F. T., et al., J. Biol. Chem. 270, 13503-13511 (1995)

SOURCE: This antibody was purified from rabbit serum
using affinity column. The rabbit was immunized with thesynthetic peptide corresponding to N-terminus of human
EEA1.

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.

kDa25012345615010075
Western blotting analysis of EEA1expression in HeLa (1), 293T (2), A549
(3), NIH/3T3 (4), MEF (5) and NRK (6)using PM062.

REACTIVITY: This antibody reacts with EEA1 forWesternblotting,Immunocytochemistry.

Immunoprecipitation

andPROTOCOLS:
SDS-PAGE & Western blotting

APPLICATIONS:
Western blotting; 1:1,000
Immunoprecipitation; 2 L/300 L of cell extract from1 x 107 cellsImmunohistochemistry; Not tested
Immunocytochemistry; 1:200
Flow cytometry; Not tested
Detailed procedures are provided in the following
PROTOCOLS.

SPECIES CROSS REACTIVITY:
Species

HumanMouse

RatCells

HeLa, 293T,
A549

NIH/3T3,
MEF

NRK

Reactivity on WB+++

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% methanol). 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 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 in
PBS] (5 minutes x 3).
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 1 hour at room temperature.
8) Wash the membrane with PBS-T (5 minutes x 3).
9) Wipe excess buffer off the membrane, and incubatemembrane with an appropriate chemiluminescencereagent for 1 minute.

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PM062
Lot 004~Page 210) 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; HeLa, 293T, A549,
NIH/3T3, MEF, NRK)

12kDa
150

EEA1

10075IgG
50Immunoprecipitation of EEA1 from HeLa
with normal rabbit IgG (1) or PM062 (2).
After immunoprecipitated with the antibody,immunocomplex was resolved on SDS-PAGE
and immunoblotted with PM062.
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 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 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 4°C. 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 4°C.
4) Centrifuge the tube at 2,500 x g for 10 seconds anddiscard the supernatant.
5) Resuspend the agarose with cold Lysis buffer.
6) Centrifuge the tube at 2,500 x g for 10 seconds anddiscard the supernatant.
7) Repeat steps 5)-6) 2-4 times.
8) 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.)

Immunocytochemistry1) Culture the cells in the appropriate condition on a glassslide. (For example, spread 1 x 104 cells for one slide,then incubate in a CO2 incubator overnight.)
2) Wash the glass slide twice 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 twice with PBS.
7) Add the primary antibody diluted with 2% FCS/PBS as
suggested in the APPLICATIONS onto the cells andincubate for 1 hour at room temperature (Optimization ofantibody concentration or incubation condition isrecommended if necessary).
8) Wash the glass slide twice with PBS.
9) Add 100 L of 1:500 Alexa Fluor® 488 conjugatedanti-rabbit IgG (Thermo Fisher Scientific, code no.
A11008) diluted with PBS onto the cells. Incubate for 30
minutes at room temperature. Keep out light by aluminum
foil.
10) Wash the glass slide 3 times with PBS.
11) Counterstain with DAPI for 5 minutes at room
temperature.
12) Wash the glass slide twice with PBS.
13) Wipe excess liquid off the slide but take care not to touchthe cells. Never leave the cells to dry.
14) Promptly add mounting medium onto the slide, then put acover slip on it.
(Positive control for Immunocytochemistry; A549)

Immunocytochemicaldetection
EEA1 in A549 with PM062.
Green: anti-EEA1
Blue: DAPI counter stainof

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(Positive control for Immunoprecipitation; HeLa)

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