Recombinant
RabMAb

Anti-muscle Actin antibody [EPR8484] (ab156302)

Overview

  • Product name
    Anti-muscle Actin antibody [EPR8484]
    See all muscle Actin primary antibodies
  • Description
    Rabbit monoclonal [EPR8484] to muscle Actin
  • Host species
    Rabbit
  • Specificity
    ab156302 will detect alpha and gamma specific actin from skeletal, cardiac and smooth muscle.
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human muscle Actin aa 350 to the C-terminus. The exact sequence is proprietary.
    Database link: P63267

  • Positive control
    • WB: HeLa, A431, A673, HEK293, MCF7, L6 and NIH 3T3 cell lysates, human fetal artery, kidney and heart tissue lysates, human uterus, stomach and skeletal muscle tissue lysates and rat spleen tissue lysates. IHC-P: Human skeletal muscle, smooth muscle in colon, cervical carcinoma and heart muscle tissues. Mouse cardiac muscle and rat colon tissues. ICC/IF: HeLa, A673 and NIH3T3 cells. Flow Cyt: HeLa cells. IP: NIH 3T3 cell lysates.
  • 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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab156302 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
IHC-P 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

WB 1/1000 - 1/10000. Predicted molecular weight: 42 kDa.
ICC/IF 1/100 - 1/250.
IP 1/10 - 1/100.
Flow Cyt 1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells.
  • Involvement in disease
    Defects in ACTA1 are the cause of nemaline myopathy type 3 (NEM3) [MIM:161800]. A form of nemaline myopathy. Nemaline myopathies are muscular disorders characterized by muscle weakness of varying severity and onset, and abnormal thread-or rod-like structures in muscle fibers on histologic examination. The phenotype at histological level is variable. Some patients present areas devoid of oxidative activity containg (cores) within myofibers. Core lesions are unstructured and poorly circumscribed.
    Defects in ACTA1 are a cause of myopathy, actin, congenital, with excess of thin myofilaments (MPCETM) [MIM:161800]. A congenital muscular disorder characterized at histological level by areas of sarcoplasm devoid of normal myofibrils and mitochondria, and replaced with dense masses of thin filaments. Central cores, rods, ragged red fibers, and necrosis are absent.
    Defects in ACTA1 are a cause of congenital myopathy with fiber-type disproportion (CFTD) [MIM:255310]; also known as congenital fiber-type disproportion myopathy (CFTDM). CFTD is a genetically heterogeneous disorder in which there is relative hypotrophy of type 1 muscle fibers compared to type 2 fibers on skeletal muscle biopsy. However, these findings are not specific and can be found in many different myopathic and neuropathic conditions.
  • Sequence similarities
    Belongs to the actin family.
  • Post-translational
    modifications
    Oxidation of Met-46 by MICALs (MICAL1, MICAL2 or MICAL3) to form methionine sulfoxide promotes actin filament depolymerization. Methionine sulfoxide is produced stereospecifically, but it is not known whether the (S)-S-oxide or the (R)-S-oxide is produced.
  • Cellular localization
    Cytoplasm > cytoskeleton.
  • Information by UniProt
  • Database links
  • Alternative names
    • a actin antibody
    • ACTA antibody
    • ACTA1 antibody
    • ACTA2 antibody
    • ACTC antibody
    • ACTC1 antibody
    • Actin antibody
    • ACTS_HUMAN antibody
    • ACTSA antibody
    • Alpha 2 actin antibody
    • alpha skeletal muscle antibody
    • Alpha-actin-1 antibody
    • Cardiac muscle alpha actin 1 antibody
    • Skeletal muscle alpha actin 1 antibody
    see all

Images

  • Overlay histogram showing HeLa cells fixed in 4% PFA and stained with purified ab156302 at a dilution of 1 in 50 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).
  • All lanes : Anti-muscle Actin antibody [EPR8484] (ab156302) at 1/10000 dilution (purified)

    Lane 1 : NIH/3T3 whole cell lysate
    Lane 2 : Rat spleen tissue lysate
    Lane 3 : L6 whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP-conjugated goat anti-rabbit IgG (specific to the non-reduced form of IgG) at 1/10000 dilution

    Predicted band size: 42 kDa
    Observed band size: 42 kDa



    Blocking and dilution buffer: 5% NFDM /TBST.

  • Anti-muscle Actin antibody [EPR8484] (ab156302) at 1/10000 dilution (purified) + Human stomach tissue lysate at 10 µg

    Secondary
    HRP-conjugated goat anti-rabbit IgG (specific to the non-reduced form of IgG) at 1/10000 dilution

    Predicted band size: 42 kDa
    Observed band size: 42 kDa



    Blocking and dilution buffer: 5% NFDM /TBST.

  • Anti-muscle Actin antibody [EPR8484] (ab156302) at 1/1000 dilution (purified) + A673 whole cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Predicted band size: 42 kDa
    Observed band size: 42 kDa



    Blocking and dilution buffer: 5% NFDM /TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat colon tissue labelling muscle Actin with purified ab156302 at a dilution of 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cardiac muscle tissue labelling muscle Actin with purified ab156302 at a dilution of 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling muscle Actin with purified ab156302 at a dilution of 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of A673 cells labelling muscle Actin with purified ab156302 at 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Flow Cytometry analysis of HeLa cells labelling muscle Actin with purified ab156302 at 1/50 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • ab156302 (purified) at 1/20 immunoprecipitating muscle Actin in NIH/3T3 whole cell lysate.

    Lane 1 (input): NIH/3T3 whole cell lysate (10µg)

    Lane 2 (+): ab156302 + NIH/3T3 whole cell lysate.

    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab156302 in NIH/3T3 whole cell lysate.

    For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/10000).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • All lanes : Anti-muscle Actin antibody [EPR8484] (ab156302) at 1/1000 dilution (unpurified)

    Lane 1 : NIH 3T3 lysate
    Lane 2 : Human fetal artery lysate
    Lane 3 : Human fetal kidney lysate
    Lane 4 : Human uterus lysate
    Lane 5 : Human stomach lysate
    Lane 6 : Human fetal heart lysate
    Lane 7 : Human skeletal muscle lysate

    Lysates/proteins at 10 µg per lane.

    Predicted band size: 42 kDa

  • All lanes : Anti-muscle Actin antibody [EPR8484] (ab156302) at 1/1000 dilution (unpurified)

    Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 5 : Skeletal Muscle (Human) Tissue Lysate - adult normal tissue
    Lane 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution

    Predicted band size: 42 kDa
    Observed band size: 42 kDa



    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab156302 overnight at 4°C. Antibody binding was detected using ab175781 at a 1/10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

    Secondary antibody - goat anti-rabbit Alexa Fluor® 790 (ab175781).

  • IHC image of unpurified ab156302 staining muscle Actin in human colon* formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab156302, 5μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labelling muscle Actin with unpurified ab156302 at a dilution of 1/1000.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human heart muscle tissue labelling muscle Actin with unpurified ab156302 at a dilution of 1/1000.

  • Unpurified ab156302 staining Actin in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with unpurified ab156302 at 10μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an goat anti-rabbit Alexa Fluor® 488 secondary (ab150081) at 2 μg/ml (shown in green) and goat anti-mouse Alexa Fluor® 594 secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

    Negative controls: 1 – Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

  • Unpurified ab156302 staining Actin in NIH3T3 cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with unpurified ab156302 at 5μg/ml and ab195889 at 1/250 dilution (shown in pseudo color red) overnight at +4°C, followed by a further incubation at room temperature for 1h with an goat anti-rabbit Alexa Fluor® 488 secondary (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Overlay histogram showing HeLa cells stained with unpurified ab156302 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab156302, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was goat anti-rabbit Alexa Fluor® 488 (IgG H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References

This product has been referenced in:

See all 4 Publications for this product

Customer reviews and Q&As

Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Reduced Denaturing (13%)
Sample
Human Cell lysate - whole cell (HCT116 and MEF cell lysate)
Specification
HCT116 and MEF cell lysate
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Christian Marx

Verified customer

Submitted Feb 21 2014

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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