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

PM060
Lot 003~
Page 1POLYCLONAL ANTIBODYER Marker

Anti-Calnexin pAb
Code No.
PM060

Quantity
100 µLBACKGROUND: 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 in
the quality control of the ER by the retention of incorrectly
folded proteins.

SOURCE: This antibody was purified from rabbit serum

using affinity column. The rabbit was immunized with thesynthetic peptide corresponding to N-terminus of humanCalnexin.

FORMULATION: 100 µL volume of PBS containing
50% glycerol, pH 7.2. Contains no preservative.

Form
Affinity Purified

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.

kDa

12

3

4150
10075

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

50

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

APPLICATIONS:

Western blotting; 1:1,000
Immunoprecipitation; 2 µL/300 µL of cell extract from1 x 107 cells
Immunohistochemistry; Not tested
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,
A549, Jurkat

NIH/3T3

Not Tested

Reactivity on WB+-REFERENCES:1) Kleizen, B., and Braakman, I., Curr. Opin. Cell Biol. 16, 343-349 (2004)
2) David, V., et al., J. Biol. Chem. 268, 9585-9592 (1993)

Western blotting analysis of Calnexin
expression on HeLa (1), 293T (2),A549 (3) and Jurkat (4) using PM060.

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 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 inPBS] (5 minutes x 3).
7) Incubate the membrane with 1:10,000 HRP-conjugatedanti-rabbit IgG (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).

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

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

PM060Lot 003~
Page 29) 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.
(Positive controls for Western blotting; HeLa, 293T, A549,Jurkat)

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°C
and transfer the supernatant to another tube.
3) Add primary antibody as suggested in theAPPLICATIONS into 300 µL of the supernatant. Mix
well and incubate with gentle agitation for 30-120 minutesat 4°C. Add 20 µL 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, andcarefully discard the supernatant using a pipettor without
disturbing the beads.
5) Resuspend the beads with cold Lysis buffer.
6) Centrifuge the tube at 2,500 x g for 10 seconds, andcarefully discard the supernatant.
7) Repeat steps 5)-6) 3-5 times8) Resuspend the beads in 20 µL of Laemmli’s sample buffer,boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10µL/lane for the SDS-PAGE analysis.
(See SDS-PAGE & Western blotting.)

Immunocytochemistry1) Culture the cells in the appropriate condition on a glass
slide. (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 2% FCS/PBS assuggested in the APPLICATIONS onto the cells andincubate for 1 hour at room temperature (Optimization ofantibody concentration or incubation condition arerecommended if necessary).
8) Wash the glass slide 2 times with PBS.
9) Add 100 µL of 1:500 Alexa Fluor® 488 conjugatedanti-rabbit IgG (Thermo Fisher Scientific, code no.
A110374) diluted with PBS onto the cells. Incubate for 30
minutes at room temperature. Keep out light by aluminumfoil.
10) Wash the glass slide 3 times with PBS.
11) Counter stain with DAPI for 5 minutes at roomtemperature.
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)

Immunocytochemical detection ofCalnexin in A549 with PM060.
Green: Anti-Calnexin pAb (MBL, code no. PM060)
Blue: DAPI counter stain
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(Positive control for Immunoprecipitation; HeLa)
231205-4