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 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) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (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).

All lanes : Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ACTA2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

Lanes 1 - 2: Merged signal (red and green). Green - ab124964 observed at 42 kDa. Red - 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Flow cytometry analysis of Jurkat (human T cell leukemia cell line from peripheral blood) cells labelling alpha smooth muscle Actin with purified ab124964 at 1/30 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

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.

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)
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, ab150113, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

All lanes : Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution (purified)

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate
Lane 3 : U937 (human histiocytic lymphoma cell line) cell lysate
Lane 4 : A431 (human epidermoid carcinoma cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

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

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/50000 dilution (purified)

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

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

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution

Lane 1 : Human lung lysate
Lane 2 : A431 (human epidermoid carcinoma cell line) cell lysate
Lane 3 : A549 (human lung carcinoma cell line) cell lysate
Lane 4 : NIH/3T3 (mouse embyro fibroblast cell line) cell lysate
Lane 5 : SV40-LT cell lysate
Lane 6 : C2C12 (mouse myoblast cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

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


Exposure time: 10 seconds

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 ab124964 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

All lanes : Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/1000 dilution (unpurified)

Lane 1 : Human heart tissue lysate - total protein (ab29431)
Lane 2 : Heart (Mouse) Tissue Lysate
Lane 3 : Heart (Rat) Tissue Lysate
Lane 4 : Human skeletal muscle tissue lysate - total protein (ab29330)
Lane 5 : HEK-293 (human epithelial cell line from embryonic kidney) Whole Cell Lysate
Lane 6 : A431 (human epidermoid carcinoma cell line) Whole Cell Lysate
Lane 7 : SV40LT-SMC (rat aorta smooth muscle) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : 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
Additional bands at: 42 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 30 seconds

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 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 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.

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 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.

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