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

PM059
Lot 004~
Page 1POLYCLONAL ANTIBODYER Marker

Anti-KDEL pAb
Code No.
PM059

Quantity
100 µLBACKGROUND: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. This antibody canspecifically detect GRP78 (78 kDa protein) and GRP94 (94kDa protein) including a KDEL sequence by WB.

SOURCE: This antibody was purified from rabbit serum

Form
Affinity Purified

REFERENCES:

1) Ishii, S., et al., Biochem. Biophys. Res. Commun. 486,767-773 (2017) [IC]2) Yamanaka, T., et al., Sci. Rep. 6, 34575 (2016) [IHC]
3) Yamanaka, T., et al., Nat. Commun. 5, 3354 (2014) [IHC]
4) Saito, A., et al., Nat. Cell Biol. 11, 1197-1204 (2009) [IHC]
5) Pelham, H. R., EMBO J. 7, 913-918 (1988)
6) Munro, S., and Pelham, H. R., Cell 48, 899-907 (1987)

using affinity column. The rabbit was immunized with theKLH conjugated synthetic peptide CTGEEDTSEKDEL
corresponding to amino acid residues 644-654 from mouse
GRP78.

FORMULATION: 100 µL 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.

REACTIVITY: This antibody reacts with GRP78 andGRP94 including KDEL sequence for Western blotting.

APPLICATIONS:

Western blotting; 1:500 for chemiluminescence detection
systemImmunoprecipitation; Not tested
Immunohistochemistry; Not tested**It is reported that this antibody can be used in
Immunohistochemistry in the reference number 2-4).
Immunocytochemistry; 1:1,000
Flow cytometry; Not tested
Detailed procedures are provided in the following
PROTOCOLS.

SPECIES CROSS REACTIVITY:
Species

Human

Mouse

Rat

Cells

HeLa, 293T

NIH/3T3

Not tested

Reactivity on WB++INTENDED USE:

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

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

PROTOCOLS:

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 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.
5) Incubate the membrane for 1 hour at room temperature
with primary antibody diluted with 1% skimmed milk (inMEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

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

PM059
Lot 004~
Page 2
PBS, 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 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 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 3 minutes. Develop the film under usual settings. Theconditions for exposure and development may vary.

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; A549)

RELATED PRODUCTS:

Please visit our website at https://ruo.mbl.co.jp/.

(Positive controls for Western blotting; HeLa, 293T andNIH/3T3)Immunocytochemistry1) 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-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 assuggested 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 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 3 times with PBS.
11) Counter stain with DAPI for 5 minutes at room221227-5