Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Lamin B1 antibody [EPR22165-121] (ab229025)

Overview

  • Product name

    Anti-Lamin B1 antibody [EPR22165-121]
    See all Lamin B1 primary antibodies
  • Description

    Rabbit monoclonal [EPR22165-121] to Lamin B1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment within Human Lamin B1 aa 450 to the C-terminus. The exact sequence is proprietary.
    Database link: P20700

  • Positive control

    • WB: HeLa, HAP1, HepG2, Caco-2, RAW 264.7, PC-12 and NIH/3T3 whole cell lysate; Human lymph node tissue; Mouse brain and heart tissue lysate; Rat brain, heart and spleen tissue lysate. IHC-P: Human breast and liver tissue; Mouse liver tissue; Rat cerebrum tissue. ICC/IF: HeLa, HAP1, and NIH/3T3 cells. Flow: HeLa, HAP1 and NIH/3T3 cells. IP: NIH/3T3 and HeLa whole cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab229025 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000. Detects a band of approximately 70 kDa (predicted molecular weight: 66 kDa).
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

ICC/IF 1/1000.
Flow Cyt 1/500.
IP 1/30.

Target

  • Function

    Lamins are components of the nuclear lamina, a fibrous layer on the nucleoplasmic side of the inner nuclear membrane, which is thought to provide a framework for the nuclear envelope and may also interact with chromatin.
  • Involvement in disease

    Defects in LMNB1 are the cause of leukodystrophy demyelinating autosomal dominant adult-onset (ADLD) [MIM:169500]. ADLD is a slowly progressive and fatal demyelinating leukodystrophy, presenting in the fourth or fifth decade of life. Clinically characterized by early autonomic abnormalities, pyramidal and cerebellar dysfunction, and symmetric demyelination of the CNS. It differs from multiple sclerosis and other demyelinating disorders in that neuropathology shows preservation of oligodendroglia in the presence of subtotal demyelination and lack of astrogliosis.
  • Sequence similarities

    Belongs to the intermediate filament family.
  • Post-translational
    modifications

    B-type lamins undergo a series of modifications, such as farnesylation and phosphorylation. Increased phosphorylation of the lamins occurs before envelope disintegration and probably plays a role in regulating lamin associations.
  • Cellular localization

    Nucleus inner membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • ADLD antibody
    • lamin B1 antibody
    • Lamin-B1 antibody
    • LMN antibody
    • LMN2 antibody
    • LMNB antibody
    • Lmnb1 antibody
    • LMNB1_HUMAN antibody
    • MGC111419 antibody
    • OTTHUMP00000159218 antibody
    see all

Images

  • Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue.

    Panel A:  Merged staining of Collagen VI (ab182744; green), anti-CD68 (ab213363; red) and anti-Lamin B1 (ab229025; magenta).

    Panel B: Anti-Collagen VI (green) stained on extracellular matrix.

    Panel C: Anti-CD68 (red) stained on Kupffer cells.

    Panel D: Anti-Lamin B1 (magenta) stained on nuclear envelope.

    Key protocol steps:  The section was incubated in three rounds of staining with ab182744 (1/1000 dilution), ab213363 (1/1000 dilution) and ab229025 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by tyramide signal amplification with the appropriate fluorophore. Heat mediated antigen retrieval was used (Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins after every round of antibody/fluorophore staining.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

    DAPI was used as a nuclear counter stain. A ready-to-use anti-Rabbit and Mouse Polymer HRP was used as a secondary.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/5000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Lamin B1 knockout HAP1 whole cell lysate
    Lane 3 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
    Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 66 kDa
    Observed band size: 70 kDa
    why is the actual band size different from the predicted?


    Exposure time: 8 seconds


    ab229025 was shown to specifically react with Lamin B1 in wild-type HAP1 cells as signal was lost in Lamin B1 knockout cells. Wild-type and Lamin B1 knockout samples were subjected to SDS-PAGE. ab229025 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Lamin B1 (Green) with ab229025 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing nuclear membranous staining in HeLa cells. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used as a counterstain at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa Fluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

     

  • Immunohistochemical analysis of paraffin-embedded mouse liver tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on mouse liver (PMID: 19925772) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Lamin B1 (Green) with ab229025 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing nuclear membranous staining in NIH/3T3 cells. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used as a counterstain at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa Fluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

     

  • All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/1000 dilution

    Lane 1 : Human lymph node tissue lysate
    Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
    Lane 4 : Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate
    Lane 5 : Mouse brain tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/100000 dilution
    Lanes 2-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 66 kDa
    Observed band size: 70 kDa why is the actual band size different from the predicted?



    Blocking/diluting buffer and concentration: 5% NFDM/TBST.

    Exposure time:

    Lane 1:3 seconds;

    Lanes 2,3,4 and 5:10 seconds.

  • All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/1000 dilution

    Lane 1 : Mouse heart tissue lysate
    Lane 2 : Rat brain tissue lysate
    Lane 3 : Rat heart tissue lysate
    Lane 4 : Rat spleen tissue lysate
    Lane 5 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
    Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
    Lane 7 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 66 kDa
    Observed band size: 70 kDa why is the actual band size different from the predicted?



    Blocking/diluting buffer and concentration: 5% NFDM/TBST.

    Exposure time:

    Lane 1:10 seconds; Lanes 2 and 3: 59 seconds; Lane 4:3 seconds; Lanes 5,6 and 7:6 seconds.

  • Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on rat cerebrum (PMID: 26469707, PMID: 19925772) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Immunohistochemical analysis of paraffin-embedded human breast tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on human breast (PMID: 26469707) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Lamin B1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab229025 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229025 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

    Lane 1: HeLa whole cell lysate 10 μg (input).
    Lane 2: ab229025 IP in HeLa whole cell lysate (+).
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229025 in HeLa whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 5 seconds.

    A very weak band around 66 kDa detected in lane 3 is due to spill over from lane2 (+).

  • Lamin B1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab229025 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229025 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

    Lane 1: NIH/3T3 whole cell lysate 10 μg (input).
    Lane 2: ab229025 IP in NIH/3T3 whole cell lysate (+).
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229025 in NIH/3T3 whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 5 seconds.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cell line labeling Lamin B1 with ab229025 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Lamin B1 with ab229025 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution.

References

ab229025 has not yet been referenced specifically in any publications.

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