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

M179-3
Lot 004~
Page 1

MONOCLONAL ANTIBODY

cis-Golgi Marker

Anti-GM130 mAb
Code No.
M179-3Clone
5G8

Subclass
Mouse IgG2a κ

BACKGROUND: The Golgi apparatus is a eukaryoticorganelle, which is mainly devoted to processing theproteins synthesized in the endoplasmic reticulum (ER).
GM130 is a member of the golgin family of coiled-coilproteins that localizes predominantly to the cis-Golgi.
GM130 might participate in ER-Golgi traffic.

SOURCE: This antibody was purified from hybridoma

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

REFERENCES:

1) Oh-hashi, K., et al., FEBS Lett. 585, 2481-7 (2011) [IC]2) Diao, A., et al., J. Biol. Chem. 283, 6957-6967 (2008)
3) Alvarez, C., et al., J. Biol. Chem. 276, 2693-2700 (2001)

kDa
250

FORMULATION: 100 µg IgG in 100 µL volume of100

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
GM130 on Western blotting, Immunoprecipitation, andImmunocytochemistry.

APPLICATIONS:
Westernblotting;

1 µg/mL for chemiluminescence
detection system
Immunoprecipitation; 2 µg/300 µL of cell extract from3 x 106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 5 µg/mL
Flow cytometry; Not tested
Detailed

procedure
PROTOCOLS.

isprovidedin

thefollowing

SPECIES CROSS REACTIVITY:
Species
Cells
Reactivity
on WB

HumanHeLa, 293T
A549

Mouse

Rat

Not tested

Not tested

Monkey*+*It is reported that this antibody can be used in COS-7 cells
in Immunocytochemistry1).

Concentration
1 mg/mL

INTENDED USE:

(clone 5G8) supernatant using protein A agarose. Thishybridoma was established by fusion of mouse myeloma
cell P3U1 with C3H mouse lymphocyte immunized withthe synthetic peptide corresponding to C-terminal of human
GM130.

PBS containing 50% glycerol, pH 7.2. No preservative iscontained.

Quantity
100 µL12315075

Western blot analysis of GM130 in
HeLa (1), 293T (2) and A549 (3) using
M179-3.
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 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 in
PBS] (5 minutes x 3 times).

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

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

M179-3
Lot 004~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 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 6 minutes. Develop the film under usual settings. Theconditions for exposure and development may vary.
(Positive controls for Western blotting; HeLa, 293T andA549)

kDa
250

12150
10075

Immunoprecipitation of GM130 fromHeLa with mouse IgG2a isotypic control,
M076-3 (1) or M179-3 (2). After
immunoprecipitated with the antibody,immunocomplexes were resolved on SDSPAGE and immunoblotted with PM061.

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 30 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
with PBS onto the cells. Incubate for 30 minutes at roomtemperature. Keep out light by aluminum foil.
10) Wash the glass slide 2 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; 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
inhibitors
atappropriate
concentrations. Incubate it at 4°C 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 fresh 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 minutes
at 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) 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 of
GM130 in HeLa using M179-3.
Green: anti-GM130
Blue: DAPI counter stain

RELATED PRODUCTS:

Please visit our web site https://ruo.mbl.co.jp/(Positive control for Immunoprecipitation; HeLa)
210720-3