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

PM061
Lot 005~
Page 1POLYCLONAL ANTIBODY

cis-Golgi Marker

Anti-GM130 pAb
Code No.
PM061

Quantity
100 LForm
Affinity Purified

BACKGROUND: The Golgi apparatus is a eukaryotic

REFERENCES:

organelle, 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.

1) Seto, S., et al. PLoS One 8, e83324 (2013) [IC]2) Tamaki, H., et al., FEBS Lett. 586, 3064-3070 (2012) [IC]3) Diao, A., et al., J. Biol. Chem. 283, 6957-6967 (2008)
4) Alvarez, C., et al., J. Biol. Chem. 276, 2693-2700 (2001)

SOURCE: This antibody was purified from rabbit serum

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

using affinity column. The rabbit was immunized with thesynthetic peptide corresponding to C-terminus of GM130.

FORMULATION: 100 L volume of PBS containing

kDa25012350% glycerol, pH 7.2. No preservative is contained.

STORAGE: This antibody solution is stable for one year

150

from the date of purchase when stored at -20°C.

REACTIVITY: This antibody reacts with human
GM130 for Western blotting, Immunoprecipitation andImmunocytochemistry.

APPLICATIONS:
Western blotting; 1:1,000
Immunoprecipitation; 2 L/300 L of cell extract from1 x 107 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1:500
Flow cytometry; Not tested
Detailed procedures are provided in the following
PROTOCOLS.

SPECIES CROSS REACTIVITY:
Species

HumanMouse*

RatCells

HeLa, 293T,A549

NIH/3T3

Not Tested

Reactivity on WB+-*This antibody does not react with mouse in Westernblotting, but can be used in Immunocytochemistry.

INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

100
Western blot analysis of GM130
expression on HeLa (1), 293T (2) andA549 (3) using PM061.
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 Lof 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% 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 (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 times).
7) Incubate the membrane with 1:10,000 of Anti-IgG(Rabbit) pAb-HRP (MBL; code no. 458) diluted with 1%
skimmed milk (in PBS, pH 7.2) for 1 hour at roomtemperature.

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PM061
Lot 005~
Page 28) 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. The
conditions for exposure and development may vary.
(Positive controls for Western blotting; HeLa, 293T, A549and Jurkat)

kDa

11502GM130

10075IgG
50
Immunoprecipitation of GM130 from HeLa
with normal rabbit IgG (1) or PM061 (2).
After immunoprecipitated with the antibody,immunocomplex was resolved on SDS-PAGE
and immunoblotted with PM061.

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-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 as
suggested 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 L 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 room
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 andNIH/3T3)

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°Cand transfer the supernatant to another tube.
3) Add primary antibody as suggested in theAPPLICATIONS into 300 L of the supernatant. Mixwell and incubate with gentle agitation for 30-120 minutes
at 4oC. Add 20 L 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 L of Laemmli’s sample buffer,boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10
L/lane for the SDS-PAGE analysis.
(See SDS-PAGE & Western blotting.)(Positive control for Immunoprecipitation; HeLa)

Immunocytochemical detection of
GM130 in A549 (upper) and in NIH/3T3(lower) with PM061.
Green: Anti-GM130 pAb (PM061)
Blue: DAPI counter stain

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