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

M178-3
Lot 005~
Page 1

MONOCLONAL ANTIBODYER Marker

Anti-Calnexin mAb
Code No.
M178-3Clone
4F10

Subclass
Mouse IgG2a κ

BACKGROUND: The endoplasmic reticulum (ER) is aeukaryotic organelle, which serves many general functions,
including the facilitation of protein folding. Calnexin is a
90 kDa integral membrane protein of the ER. Calnexin is
one of the chaperone proteins, which play a major role inthe quality control of the ER by the retention of incorrectly
folded proteins.

SOURCE: This antibody was purified from hybridoma(clone 4F10) supernatant using protein A agarose. Thishybridoma was established by fusion of mouse myeloma
cell P3U1 with C3H mouse lymphocyte immunized withsynthetic peptide corresponding to N-terminus of humanCalnexin.

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.

REACTIVITY: This antibody reacts with humanCalnexin for Western blotting, Immunoprecipitation andImmunocytochemistry.

APPLICATIONS:

Western blotting; 0.1 µg/mL
Immunoprecipitation; 1 µg/300 µL of cell extract from 3 x
106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1 µg/mL
Flow cytometry; Not tested
Detailed procedures are provided in the following
PROTOCOLS.

SPECIES CROSS REACTIVITY:
Species
Cells
Reactivity
on WB

HumanHeLa, 293T,
A549, Jurkat+Mouse
NIH/3T3

Rat

MonkeyNot tested Not tested*-*Reactivity of clone 4F10 to monkey is not confirmed inour laboratory. However, it is reported that this clone
reacts with COS-7 cells2).

Quantity
100 µL

Concentration
1 mg/mL

REFERENCES:

1) Matsuzaka, Y., et al., PLoS One 11, e0167811 (2016) [WB]
2) Oh-hashi, K., et al., FEBS Lett. 585, 2481-2487 (2011) [IC]3) Kleizen, B. and Braakman, I., Curr. Opin. Cell Biol. 16,343-349 (2004)
4) David, V., et al., J. Biol. Chem. 268, 9585-9592 (1993)

INTENDED USE:

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

kDa15012341007550
Western blot analysis of Calnexin in
HeLa (1), 293T (2), A549 (3) and Jurkat(4) using M178-3.
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 3 minutes and centrifuge. Load 20 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 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 5%
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 inPBS] (5 minutes x 3).

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

M178-3
Lot 005~
Page 27) 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).
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 1 minute. Develop the film under usual settings. The
conditions for exposure and development may vary.
(Positive controls for Western blotting; HeLa, 293T, A549,Jurkat)

kDa

12100

Calnexin

IgG Heavy chain

Immunoprecipitation of Calnexin fromHeLa with mouse IgG2a isotype control,
M076-3 (1) or M178-3 (2). After
immunoprecipitated with the antibody,immunocomplexes were resolved on SDSPAGE and immunoblotted with M178-3.

Immunoprecipitation

Immunocytochemical detection ofCalnexin in HeLa using M178-3.

Green: anti-Calnexin
Blue: DAPI counter stain

Immunocytochemistry7550(Positive control for Immunoprecipitation; HeLa)

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 at appropriate concentrations.
Incubate it at 4°C with rotating for 30 minutes; thereafter,briefly sonicate the mixture (up to 10 seconds).
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 4°C. 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) 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 times8) 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,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-100for 10 minutes at room temperature.
6) Wash the glass slide twice with PBS.
7) Add the primary antibody diluted with PBS as suggested
in the APPLICATIONS onto the cells and incubate for 1hour at room temperature (Optimization of antibody
concentration or incubation condition is recommended ifnecessary).
8) Wash the glass slide twice with PBS.
9) Add 100 µL of 1:500 Alexa Fluor® 488 conjugatedanti-mouse IgG (Thermo Fisher Scientific, code no.
A-11001) 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) Counter stain with DAPI for 5 minutes at roomtemperature.
12) Wash the glass slide twice 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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