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

PM063
Lot 004~
Page 1POLYCLONAL ANTIBODY

Mitochondrial Marker

Anti-COX4 pAb
Code No.
PM063

Quantity
100 µLBACKGROUND:

The
mitochondria
aremembrane-enclosed organelle found in most eukaryoticcells. The mitochondria serve many general functions,including production of ATP, signaling, cellulardifferentiation, cell death, as well as the control of the cell
cycle and cell growth.
COX4 is the nuclear-encoded subunit IV isoform 1 of themitochondrial respiratory chain enzyme. This antibody isan effective mitochondrial marker, because COX4 isconstantly expressed at high level in mitochondria.

SOURCE: This antibody was purified from rabbit serum

using affinity column. The rabbit was immunized withsynthetic peptide corresponding to N-terminus of human
COX4.

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 human COX4on
Westernblotting,
Immunoprecipitation,andImmunocytochemistry. The reactivity to mouse, rat,
hamster and chicken COX4 was confirmed by Westernblotting.

FormAffinity Purified

INTENDED USE:

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

REFERENCES:

1) Van Kuilenburg, A. B., et al., Biochim. Biophys. Acta
1119, 218-224 (1992)
2) Zeviani, M., et al., Gene 55, 205-217 (1987)

kDa 123456737
25
2015

COX4

Western blot analysis of COX4 inHeLa (1), 293T (2), NIH/3T3 (3), MEF(4), Rat1 (5), CHO (6) and MuH1 (7)using PM063.
The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

PROTOCOLS:

SDS-PAGE & Western Blotting

APPLICATIONS:

Western blotting; 1:1,000 for a chemiluminescence
detection system
Immunoprecipitation; 2 µL/300 µL of cell extract from3 x 106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1:500
Flow cytometry; Not tested
Detailed procedures are provided in the following
PROTOCOLS.

SPECIES CROSS REACTIVITY:
Species

Human

Mouse

Rat

Hamster

Chicken

Cells

HeLa
293T

NIH/3T3
MEF

Rat1

CHO

MuH1

Reactivity on
WB+++++1) 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 roomtemperature, 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).

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

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

PM063
Lot 004~Page 27) 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 1 minute. Develop the film under usual settings. The
conditions for exposure and development may vary.
(Positive controls for Western blotting; HeLa, 293T,NIH/3T3, MEF, Rat1, CHO and MuH1)

kDa 1 25037IgG heavy chain

25
20
15

COX4

Immunoprecipitation of COX4 fromHeLa with normal rabbit IgG (1) orPM063 (2). After immunoprecipitated withthe antibody, immunocomplexes wereresolvedonSDS-PAGEandimmunoblotted with PM063.

Immunocytochemistry1) Culture the cells in the appropriate condition on a glassslide. (for example, spread 1x104 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) The glass slide was washed with PBS 3 times.
5) Immerse the slide in PBS containing 0.2% TritonX-100for 10 minutes at room temperature.
6) The glass slide was washed 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) The glass slide was washed 2 times with PBS.
9) Add 100 µL of 1:500 Alexa Fluor®488 conjugatedanti-rabbit IgG (Invitrogen; code no. A110374) diluted
with PBS onto the cells. Incubate for 30 minutes at roomtemperature. Keep out light by aluminum foil.
10) The glass slide was washed 2times with PBS.
11) Counter stain with DAPI for 5 minutes at room
temperature.
12) The glass slide was washed 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)

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
inhibitorsatappropriate
concentrations. Incubate it at 4oC with rotating for 30minutes; thereafter, briefly sonicate the mixture (up to 20seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°Cand 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 room temperature. 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 roomtemperature.
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 10µL/lane for the SDS-PAGE analysis.
(See SDS-PAGE & Western blotting.)

Immunocytochemical detection ofCOX4 in HeLa using PM063.

Green: anti-COX4
Blue: DAPI counter stain

RELATED PRODUCTS:

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

220118-3