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.

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).

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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

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.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

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.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

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 (K_D)
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