Anti-alpha smooth muscle Actin antibody [EPR5368]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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, ab150081) secondary antibody used at a 1/5000 dilution.

Isotype control : Rabbit monoclonal IgG (ab172730) black line.

Unlabelled control : Cells without incubation with primary antibody and secondary antibody blue line.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

AbReview62598****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

This image is courtesy of an anonymous Abreview.

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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

Secondary

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

false

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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

false

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

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

Lane 2:

Western blot - Human ACTA2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-acta2-knockout-hela-cell-line-ab264014'>ab264014</a>)

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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

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

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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 (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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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 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).

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

All lanes:

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/1000 dilution

Lane 1:

Human heart tissue lysate - total protein (<a href='/en-us/products/unavailable/human-heart-tissue-lysate-total-protein-ab29431'>ab29431</a>) 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 (<a href='/en-us/products/unavailable/human-skeletal-muscle-tissue-lysate-total-protein-ab29330'>ab29330</a>) 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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

true

Exposure time: 30s

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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:

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

Predicted band size: 42 kDa

Observed band size: 42 kDa

true

Exposure time: 10s

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-alpha smooth muscle Actin antibody [EPR5368] (AB124964)

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.