Anti-Actin antibody [EPR16769] - Loading Control

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Actin with purified ab179467 at 1/70 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelia cell) cells labeling Actin with Purified ab179467 at 1 : 100 dilution ( 6.98 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunohistochemical analysis of formalin fixed paraffin embedded human skeletal muscle labelling Actin with ab179467 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab179467 anti-Actin antibody [EPR16769] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunohistochemical analysis of paraffin-embedded Human prostate hyperplasia tissue labeling Actin with ab179467 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining on smooth muscle cells is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling Actin with ab179467 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling Actin with ab179467 at 1/50 dilution, followed by Goat anti-rabbit Alexa Fluor^® 488 (IgG) (ab150077) secondary antibody at 1/200 dilution (green). Cytoplasm staining on NIH/3T3 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse Alexa Fluor^® 594 secondary) at 1/500 dilution (red).

The negative controls are as follows;
1. ab179467 at 1/50 dilution followed by ab150120 (goat anti-mouse Alexa Fluor^® 594 secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (goat anti-rabbit Alexa Fluor^® 488 (IgG H&L) at 1/200 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling Actin with Purified ab179467 at 1 : 100 dilution (6.98 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling Actin with ab179467 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Actin was immunoprecipitated fromC6 (rat glial tumor glial cell) whole cell lysate with ab179467 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab179467 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Actin antibody [EPR16769] - Loading Control (ab179467) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate (Input) at 10 µg

Lane 2:

C6 (rat glial tumor glial cell) whole cell lysate (+)

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab179467 in C6 whole cell lysate (-)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 42 kDa

false

Exposure time: 6s

• IP

Supplier Data

Immunoprecipitation - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Actin was immunoprecipitated from 1mg of NIH/3T3 (Mouse embyro fibroblast cells) whole cell extract with ab179467 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab179467 at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as secondary antibody at 1/1000 dilution.

Lane 1 : NIH/3T3 whole cell extract.
Lane 2 : PBS instead of NIH/3T3 whole cell extract.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Actin antibody [EPR16769] - Loading Control (ab179467)

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (ab179467) at 1/5000 dilution

Lane 1:

Human fetal brain tissue lysate at 10 µg

Lane 2:

Human fetal heart tissue lysate at 10 µg

Lane 3:

Human fetal kidney tissue lysate at 10 µg

Lane 4:

Human fetal spleen tissue lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (ab179467) at 1/20000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates at 20 µg

Lane 2:

293T (Human epithelial cells from embryonic kidney) whole cell lysates at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Lane 4:

Human fetal spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (ab179467) at 1/20000 dilution

Lane 1:

UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) whole cell lysates) at 10 µg

Lane 2:

Human cardiac muscle tissue lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (ab179467) at 1/20000 dilution

Lane 1:

Mouse brain tissue lysate at 10 µg

Lane 2:

Mouse heart tissue lysate at 10 µg

Lane 3:

Mouse kidney tissue lysate at 10 µg

Lane 4:

Mouse spleen tissue lysate at 10 µg

Lane 5:

Rat brain tissue lysate at 10 µg

Lane 6:

Rat heart tissue lysate at 10 µg

Lane 7:

Rat kidney tissue lysate at 10 µg

Lane 8:

Rat spleen tissue lysate at 10 µg

Lane 9:

C6 (Rat glial tumor cells) whole cell lysates at 10 µg

Lane 10:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 11:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 12:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Actin antibody [EPR16769] - Loading Control (AB179467)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression of Ref2P is upregulated in response to reducing Atg8a (PMID : 18347073).

Loading control : Anti-Actin antibody [EPR16769] (ab179467) 1 : 5000 (42Kda).

All lanes:

Western blot - Anti-Ref2P antibody [EPR28159-511] (<a href='/en-us/products/primary-antibodies/ref2p-antibody-epr28159-511-ab323859'>ab323859</a>) at 1/1000 dilution

Lane 1:

SL2 (Drosophila melanogaster embryo epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

SL2 transfected with siRNA specifically targeting Atg8a whole cell lysate at 20 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 1/100000 dilution

Observed band size: 110 kDa,42 kDa

false

Exposure time: 180s