Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells labeling alpha smooth muscle Actin (green) with purified ab32575 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counterstained with ab7291, anti-Tubulin (mouse mAb) at 1/1000 followed by ab150120 Alexa Fluor^®594 goat anti-mouse secondary (1/1000). Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and anti-mouse secondary antibody (ab150120) were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Paraffin embedded human stomach tissue slides were dewaxed, followed by heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, Epitope Retrieval ER2 Solution) for 20 mins. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 mins. The sections were then incubated with ab32575 (1/2000) at room temperature for 30 mins, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The sections were counterstained with Hematoxylin. The images were taken with a Leica Aperio AT2.

ab32575 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in IHC-P. The image shows the staining intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human smooth muscle tissue labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/200. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, an 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/20 (red).

Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies.

Alexa Fluorr^®488 (ab197240) and Alexa Fluorr^®647 (ab196919) conjugated versions are available for this clone.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3(Mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/500 dilution (5.2 © : g/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor� 594) 1/200 (2.5 © : g/ml). Goat anti rabbit IgG (Alexa Fluor� 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 © : g/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Immunocytochemistry/ Immunofluorescence analysis of C6(Rat glial tumor glial cell) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/100 dilution (0.71 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse smooth muscle tissue labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/200. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, an 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.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

FoxA1 (red) and alpha smooth muscle actin (green) staining are shown by indirect immunofluorescence on sections of prostate from mice of the indicated genotypes.

Wild type : 21 weeks, Tgfbr2^r/r : 44 weeks, Pten^r/r : 21 weeks, Pten^r/r;Tgfbr2^r/r : 11 weeks, Apc^r/r : 36 weeks, and Apc^r/r;Tgfbr2^r/r : 24 weeks old.

IF images were captured on an Olympus BX51 microscope and DP70 digital camera, or on a Nikon Eclipse NI-U and captured with a DS-QI1 camera with NIS Elements software.

Image from Bjerke GA. et al PLoS One. 2014 Mar 20;9(3):e92800. doi: 10.1371/journal.pone.0092800. eCollection 2014.

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of MCF7 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab32575 (1/5000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab32575 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575) at 1/5000 dilution

All lanes:

MCF7 whole cell lysate at 15 µg with NDFM/TBST

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 1/20000 dilution

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575) at 1/5000 dilution

Lane 1:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 42 kDa

false

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Different batches of ab32575 were tested on A431 (Human epidermoid carcinoma epithelial cell) lysate at 0.005 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 (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575)

false

• WB

Lab

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575) at 1/5000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 42 kDa

false

• ELISA

Supplier Data

ELISA - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Following reconstitution in PBS with 10% DMSO, peptides (1 ug per mL) were immobilised in PBS on an ELISA plate overnight. After blocking in 5% BSA, primary antibody (ab271180) was added in a concentration range of 0.017-1000 ng per mL.

Pre-adsorbed secondary antibody goat anti-rabbit IgG H&L (HRP, ab97080) was used at 1/20000 dilution.

• ELISA

Supplier Data

ELISA - Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (AB32575)

Following reconstitution in PBS with 10% DMSO, peptides (1 ug per mL) were immobilised in PBS on an ELISA plate overnight. After blocking in 5% BSA, primary antibody (ab271180) was added in a concentration range of 0.017-1000 ng per mL.

Pre-adsorbed secondary antibody goat anti-rabbit IgG H&L (HRP, ab97080) was used at 1/20000 dilution.