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

M176-3
Lot 005~Page 1

MONOCLONAL ANTIBODY

Early Endosome Marker

Anti-EEA1 mAb
Code No.
M176-3Clone
3C10Subclass
Mouse IgG2a κ

BACKGROUND: The early endosome is a cellularcompartment 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 isrequired for vesicular transport of proteins through earlyendosomes. It binds membrane lipids through its FYVEdomain.

Quantity100 µL

Concentration
1 mg/mL

SPECIES CROSS REACTIVITY:
Species

HumanMouse

RatCells

HeLa, A549

NIH/3T3,
MEF

NRKReactivity on WB+++

SOURCE: This antibody was purified from hybridoma(clone 3C10) supernatant using protein A agarose. Thishybridoma was established by fusion of mouse myeloma
cell P3U1 with C3H mouse lymphocyte immunized withthe synthetic peptide corresponding to N-terminal of humanΕΕΑ1.

FORMULATION: 100 µg IgG in 100 µL volume ofPBS containing 50% glycerol, pH 7.2. No preservative iscontained.

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

The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

REACTIVITY: This antibody reacts with human ΕΕΑ1PROTOCOLS:

on
Westernblotting,Immunoprecipitation,andImmunocytochemistry. The reactivity to mouse and rat
EEA1 was confirmed by Western blotting.

APPLICATIONS:
Westernblotting;

1 µg/mL for chemiluminescence
detection system
Immunoprecipitation; 2 µg/200 µL of cell extract from3 x 106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1 µg/mL
Flow cytometry; Not tested
Detailed

procedure
PROTOCOLS.

isprovided

inthefollowing

INTENDED USE:

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

REFERENCES:

1) Gaullier, J. M., et al., J. Biol. Chem. 275, 24595-24600 (2000)
2) Mu, F. T., et al., J. Biol. Chem. 270, 13503-13511 (1995)

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 3 minutes and centrifuge. Load 10µL of the sample per lane in 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 of Anti-IgG(Mouse) pAb-HRP (MBL; code no. 330) diluted with 1%MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

URL https://ruo.mbl.co.jp e-mail support@mbl.co.jp

M176-3
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skimmed milk (in PBS, pH 7.2) for 1 hour at roomtemperature.
8) Wash the membrane with PBS-T (5 minutes x 3 times).
9) Wipe excess buffer off the membrane, and incubate
membrane with appropriate chemiluminescence reagent
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. Theconditions for exposure and development may vary.
(Positive controls for Western blotting; HeLa, A549,
NIH/3T3, MEF and NRK)

Immunocytochemistry

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 30
minutes; thereafter, briefly sonicate the mixture (up to 10seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°Cand transfer the supernatant to another fresh tube.
3) Add primary antibody as suggested in theAPPLICATIONS into 300 µL of the supernatant. Mix
well and incubate with gentle agitation for 60-120 minutes
at 4oC. Add 20 µL of 50% protein A agarose beads
resuspended in the cold IP buffer [10 mM Tris-HCl (pH8.0), 500 mM NaCl, 0.1% NP-40]. Mix well and incubatewith gentle agitation for 60 minutes at 4°C.
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 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.)

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 30 minutes 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 200 µL of 1:500 Alexa Fluor® 488 conjugatedanti-mouse IgG (Invitrogen; code no. A11001) 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) Counter stain with DAPI for 5 minutes at room
temperature.
12) Wash the glass slide 2 times with PBS.
13) Wipe excess liquid off the slide but take care not to touch
the 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; HeLa)

RELATED PRODUCTS:
Please visit our web site https://ruo.mbl.co.jp/(Positive control for Immunoprecipitation; HeLa)
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