M181-3
Lot 005~
Page 1For Research Use Only.
Not for use in diagnostic procedures.

MONOCLONAL ANTIBODYER Marker

Anti-KDEL mAb
Code No.
M181-3Clone
1D5

Subclass
Mouse IgG2a κ

BACKGROUND:The KDEL (lys-asp-glu-leu)sequence is most common endoplasmic reticulum (ER)retention signal. The ER resident proteins, which should belocated in the ER, have this retention motif, and thisretention is mediated by KDEL receptor.

SOURCE: This antibody was purified from hybridoma(clone 1D5) supernatant using protein A agarose. Thishybridoma was established by fusion of mouse myeloma
cell P3U1 with C3H mouse lymphocyte immunized withKLH conjugated synthetic peptide CTGEEDTSEKDEL
corresponding to the amino acid residues 644-655 of mouse
GRP78.

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

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

REFERENCES:

1) van de Hoef, D. L., et al., Development 140, 810-819 (2013) [IC]2) Furuichi, T., et al., Mamm Genome. 22, 318-28 (2011) [IHC]
3) Zhang, B., et al., Blood 118, 3384-91 (2011) [IC]4) Xiang, Y., et al., PLoS One 5, e13820 (2010) [WB, IP, IC]
5) Hino, S., et al., J. Bone Miner. Metab. 28, 131-138 (2010) [IHC]
6) Pelham, H. R., EMBO J. 7, 913-918 (1988)
7) Munro, S., et al., Cell 13, 899-907 (1987)

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

kDa
100

REACTIVITY: This antibody reacts with human

75

APPLICATIONS:

procedurePROTOCOLS.

isprovided

infollowing

Others*SPECIES CROSS REACTIVITY:
HumanMouse

Rat

Hamster

Cells

HeLa,293T

NIH/3T3

Rat1

CHO++++Reactivity
on WB

23450

5

Proteins
containing
KDEL
sequence

PROTOCOLS:

theSpecies

1

Western blot analysis of proteins
containing KDEL sequence in HeLa
(1), 293T (2), NIH/3T3 (3), Rat1 (4) andCHO (5) using M181-3.

Western blotting; 0.1 µg/mL
Bands may be detected at 48, 60, 78, 94 kDa.
Immunoprecipitation; Reference 4)
Immunohistochemistry; Reference 2) and 5)
Immunocytochemistry; 0.5 µg/mL
Flow cytometry; Not tested

Detailed

Concentration
1 mg/mL

INTENDED USE:

from the date of purchase when stored at -20°C.

proteins containing KDEL sequence on Western blottingand Immunocytochemistry. The reactivity to mouse, rat andhamster proteins containing KDEL sequence wasconfirmed by Western blotting.

Quantity
100 µL*Reactivity of this clone to Drosophila Txnip/VDUP1 isdescribed in the reference number 1).

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 10 µLof 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 room
temperature, or overnight at 4°C.

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M181-3Lot 005~Page 25) 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 willdepend on the conditions.)
6) Wash the membrane with PBS-T [0.05% Tween-20 in
PBS] (5 minutes x 3 times).
7) Incubate the membrane with 1:10,000 anti-IgG (Mouse)
pAb-HRP (MBL; code no. 330) 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 30 seconds. Develop the film under usual settings. The
conditions for exposure and development may vary.

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) 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)

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(Positive controls for Western blotting; HeLa, 293T,
NIH/3T3, Rat1, CHO)

Immunocytochemical detection ofproteins
containing
KDEL
sequence in HeLa using M181-3.
Green: anti-KDEL
Blue: DAPI counter stain

Immunocytochemistry1) Culture the cells in the appropriate condition on a glassslide. (for example, spread 1 x 104 cells of HeLa cells forone slide, then incubate in a CO2 incubator for one night.)
2) Wash the cells 3 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-100for 10 minutes at room temperature.
6) Wash the glass slide 2 times with PBS.
7) Add the primary antibody diluted with 2% FCS/PBS as
suggested in the APPLICATIONS onto the cells andincubate for 60 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 100 µL of 1:500 Alexa Fluor®488 conjugatedanti-mouse IgG (Invitrogen; code no. 53818A) diluted
240123-3