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

PM064
Lot 006~Page 1

POLYCLONAL ANTIBODY

Nuclear Envelope Marker

Anti-Lamin B1 pAb
Code No.
PM064

Quantity
100 LForm
Affinity Purified

BACKGROUND: Lamin B1 is one of the component

REFERENCES:

proteins of the nuclear lamina that is a dense fibrous
network and lies on the inner surface of the inner nuclear
membrane. The nuclear lamina plays an essential role inchromatin organization, cell cycle regulation, DNAreplication, cell differentiation and apoptosis.
This Lamin B1 polyclonal antibody is an effective nuclear
envelope marker for Immunocytochemistry, Western
blotting and Immunoprecipitation.

1) Bai, D., et al., Nat. Commun. 7, 12310 (2016) [WB]
2) Qu, L., et al., J. Mol. Cell Biol. 7, 529-542 (2015) [WB]3) Mukai, R. and Ohshima, T., Oncogene 33, 2317-2328 (2014) [WB]
4) Yamamoto, N., et al., Virus Res. 178, 404-410 (2013) [WB]
5) Jia, X., et al., Dig. Dis. Sci. 58, 2212-2222 (2013) [WB]
6) Jia, X., et al., Life Sci. 90, 934-943 (2012) [WB]
7) Gruenbaum, Y., et al., J. Struct. Biol. 129, 313-323 (2000)
8) Lin, F., et al., Genomics 27, 230-236 (1995)

SOURCE: This antibody was purified from rabbit serumkDa

by affinity column chromatography. The rabbit wasimmunized with KLH conjugated synthetic peptide
corresponding to C-terminus of human Lamin B1.

15010075123456Lamin B150

FORMULATION: 100 L volume of PBS containing

3750% glycerol, pH 7.2. No preservative is contained.

25

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 Lamin
B1 on Western blotting, Immunoprecipitation and
Immunocytochemistry. The reactivity to mouse, rat andhamster Lamin B1 was confirmed by Western blotting.

APPLICATIONS:
Western blotting; 1:1,000 for a chemiluminescence
detection system
Immunoprecipitation; 5 L/300 L of cell extract from3 x 106 cells
Immunohistochemistry; Not tested
Immunocytochemistry; 1:200
Flow cytometry; Not tested
Detailed

procedures

areprovided

inthefollowing

PROTOCOLS.
SPECIES CROSS REACTIVITY:
Species

HumanMouse

RatHamster

CellsHeLa
293T

NIH/3T3MEF

Rat1

CHO

Reactivity on
WB++++INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

Western blot analysis of Lamin B1 inHeLa (1), 293T (2), NIH/3T3 (3), MEF(4), Rat1 (5) and CHO (6) using
PM064.
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 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 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).

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URL https://ruo.mbl.co.jp

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PM064
Lot 006~
Page 2
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 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 and CHO)

kDa 1

Immunocytochemistry

21007550

Immunocytochemical detection ofLamin B1 in HeLa using PM064.
Green: anti-Lamin B1
Red: anti--Tubulin (M175-3)

Lamin B1
IgG heavy chain

37

Immunoprecipitation of Lamin B1 fromHeLa with normal rabbit IgG (1) or
PM064 (2). After immunoprecipitated withthe antibody, immunocomplexes wereresolvedonSDS-PAGEandimmunoblotted with PM064.
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 20seconds).
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 L of the supernatant. Mixwell and incubate with gentle agitation for 30-120 minutesat room temperature. 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 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 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.)

1) 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-100
for 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 is recommended if necessary.)8) The glass slide was washed 2 times with PBS.
9) Add 200 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) The glass slide was washed 2 times with PBS.
11) Wipe excess liquid off the slide but take care not to touch
the cells. Never leave the cells to dry.
12) Promptly add mounting medium onto the slide, then put acover slip on it.
(Positive control for Immunocytochemistry; HeLa)

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(Positive control for Immunoprecipitation; HeLa)
230328-4