Anti-alpha smooth muscle Actin antibody [EPR5368] ab124964 is a rabbit monoclonal antibody that is used in alpha smooth muscle Actin western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with ACTA2 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR5368 is cited in over 570 publications
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
IHC-P | WB | ICC/IF | Flow Cyt (Intra) | |
---|---|---|---|---|
Human | Tested | Tested | Tested | Tested |
Mouse | Tested | Tested | Expected | Expected |
Rat | Expected | Tested | Expected | Expected |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/1000 - 1/2500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Human | Dilution info 1/1000 - 1/2500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/10000 - 1/50000 | Notes For unpurified use at 1/1000. |
Species Rat | Dilution info 1/10000 - 1/50000 | Notes For unpurified use at 1/1000. |
Species Human | Dilution info 1/10000 - 1/50000 | Notes For unpurified use at 1/1000. |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/250 - 1/500 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/10 - 1/100 | Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
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Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells.
ACTSA, ACTVS, GIG46, ACTA2, Alpha-actin-2, Cell growth-inhibiting gene 46 protein
Anti-alpha smooth muscle Actin antibody [EPR5368] ab124964 is a rabbit monoclonal antibody that is used in alpha smooth muscle Actin western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with ACTA2 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR5368 is cited in over 570 publications
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EPR5368
Affinity purification Protein A
2.2 x 10-11 M
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
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 monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
This supplementary information is collated from multiple sources and compiled automatically.
Alpha smooth muscle actin (α-SMA) also known as ACTA2 is an actin isoform with a specific role in the contractile function of smooth muscle cells. The molecular weight of α-SMA is approximately 42 kDa. This protein is expressed widely in vascular smooth muscle cells the peritoneal lining and myofibroblasts. It often serves as a marker for these cell types. The expression of α-SMA is critical for the mechanical attributes of cells contributing to the rigidity and contractility of tissues where it is present.
Alpha smooth muscle actin aids in maintaining the structural integrity of tissues by forming part of the actin cytoskeleton an important element in cellular support. It exists in high concentration in stress fibers contributing to cellular movements and shape maintenance. These actions are essential in various dynamic cellular processes such as cell migration and adhesion. Alpha smooth muscle actin does not typically form complexes but it associates with other components of the actin cytoskeleton to ensure cell stability and function.
Alpha smooth muscle actin functions significantly within the TGF-beta signaling pathway which influences cell proliferation differentiation and apoptosis. It interacts closely with proteins such as myosin to facilitate cellular contractility and motility. Additionally α-SMA plays a part in the RhoA/Rho kinase (ROCK) pathway connecting with regulators of actin filament organization. These pathways are essential for modulation of smooth muscle contraction and actin filament assembly contributing to vascular development and wound healing.
Alpha smooth muscle actin is importantly involved in fibrotic diseases and vascular diseases like arteriosclerosis. Increased expression of α-SMA is often observed in fibrotic tissues contributing to pathogenesis due to excessive extracellular matrix deposition. In the context of vascular disorders overexpression of α-SMA can lead to abnormal vascular remodeling often seen in diseases like hypertension. Interactions with proteins such as fibronectin and collagens facilitate these pathological changes highlighting the importance of α-SMA in disease development.
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This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
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ab124964 staining alpha smooth muscle Actin in SV40LT-SMC cells (positive control, top panel) and A431 cells (negative control, bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab124964 at 0.5μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse stomach tissue labelling alpha smooth muscl Actin with ab124964 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.
Positive staining on smooth muscle in mouse stomach.
ab124964 staining alpha smooth muscle Actin in Mouse Uterus tissue sections by Immunohistochemistry (Formalin/PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, blocked with PB Protein Block ab64226 for 10 minutes at Room temperature and antigen retrieval was by heat mediation in citrate buffer. The sample was incubated with primary antibody (1/2000) for 30 minutes. A HRP-conjugated Goat anti-rabbit polyclonal (undiluted) was used as the secondary antibody.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labelling alpha smooth muscl Actin with purified ab124964 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.
ab124964 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab124964 at 1/500 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Lanes 1 - 2: Merged signal (red and green). Green - ab124964 observed at 42 kDa. Red - loading control, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab124964 was shown to react with alpha smooth muscle Actin in wild-type HeLa cells in western blot. Loss of signal was observed when ACTA2 knockout sample was used. Wild-type HeLa and ACTA2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab124964 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 10000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: ACTA2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 42 kDa
Observed band size: 42 kDa
Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling alpha smooth muscle Actin with ab124964 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% tritonX-100. An Goat anti rabbit IgG(Alexa Fluor® 488)
Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.
Immunocytochemistry/Immunofluorescence analysis of A-673 (human muscle Ewing's Sarcoma cell line) cells labelling alpha smooth muscle Actin with purified ab124964 at 1/300. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.
Control: primary antibody (1/300) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) ab150113, an Alexa Fluor® 488-conjugated goat anti-mouse IgG (1/500).
Different batches of ab124964 were tested on HEK-293 (Human embryonic kidney epithelial cell) lysate at 2.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 42 kDa.
All lanes: Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964)
Predicted band size: 42 kDa
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
All lanes: Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution
Lane 1: HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg
Lane 2: HEK-293 (human epithelial cell line from embryonic kidney) cell lysate at 20 µg
Lane 3: U937 (human histiocytic lymphoma cell line) cell lysate at 20 µg
Lane 4: A431 (human epidermoid carcinoma cell line) cell lysate at 20 µg
All lanes: Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 42 kDa, 45 kDa, 75 kDa
Observed band size: 42 kDa, 45 kDa, 60-72 kDa
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
All lanes: Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/50000 dilution
Lane 1: Mouse brain tissue lysate at 20 µg
Lane 2: Rat brain tissue lysate at 20 µg
All lanes: Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 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 2% milk before being incubated with abX overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406
All lanes: Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution
Lane 1: Human lung lysate at 20 µg
Lane 2: A431 (human epidermoid carcinoma cell line) cell lysate at 20 µg
Lane 3: A549 (human lung carcinoma cell line) cell lysate at 20 µg
Lane 4: NIH/3T3 (mouse embyro fibroblast cell line) cell lysate at 20 µg
Lane 5: SV40-LT cell lysate at 20 µg
Lane 6: C2C12 (mouse myoblast cell line) cell lysate at 20 µg
Developed using the ECL technique.
Predicted band size: 42 kDa
Observed band size: 42 kDa
Exposure time: 10s
All lanes: Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/1000 dilution
Lane 1: Human heart tissue lysate - total protein (ab29431) at 10 µg
Lane 2: Heart (Mouse) Tissue Lysate at 10 µg
Lane 3: Heart (Rat) Tissue Lysate at 10 µg
Lane 4: Human skeletal muscle tissue lysate - total protein (ab29330) at 10 µg
Lane 5: HEK-293 (human epithelial cell line from embryonic kidney) Whole Cell Lysate at 10 µg
Lane 6: A431 (human epidermoid carcinoma cell line) Whole Cell Lysate at 10 µg
Lane 7: SV40LT-SMC (rat aorta smooth muscle) Whole Cell Lysate at 10 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 42 kDa
Observed band size: 42 kDa
Exposure time: 30s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human heart tissue labelling alpha smooth muscle actin with unpurified ab124964 at 1/1000 dilution. Note positive staining on smooth muscle cells but negative on striated muscle cells.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human liver vessels tissue labelling alpha smooth muscle Actin with unpurified ab124964.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human colon smooth muscle tissue labelling alpha smooth muscle Actin with unpurified ab124964.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostatic carcinoma smooth muscles tissue labelling alpha smooth muscle Actin with unpurified ab124964.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis showing vascular smooth muscle staining in skeletal muscle tissue using alpha smooth muscle Actin with unpurified ab124964. Note positive staining on smooth muscle cells but negative on striated muscle cells.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovary tissue labelling alpha smooth muscle Actin with unpurified ab124964 at 1/1000 dilution.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach carcinoma smooth muscles tissue labelling alpha smooth muscle Actin with unpurified ab124964.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human tonsil vessels tissue labelling alpha smooth muscle Actin with unpurified ab124964.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.
Tissue Microarrays stained for Anti-alpha smooth muscle Actin antibody [EPR5368] using ab124964 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The section was incubated with ab124964 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
Flow cytometry overlay histogram showing Jurkat cells (human T cell leukemia T lymphocyte from peripheral blood) fixed with 4% paraformaldehyde and permeabilised with 90% methanol, stained with ab124964 at 1/1000 dilution (0.05 μg) red line. Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody used at a 1/5000 dilution.
Isotype control: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) black line.
Unlabelled control: Cells without incubation with primary antibody and secondary antibody blue line.
Immunohistochemical analysis of formalin-fixed paraffin-embedded human endometrium labelling alpha smooth muscle actin with ab124964 at a concentration of 0.07µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab124964 anti alpha smooth muscle actin antibody was incubated at 37°C for 16min. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
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