ab8226 staining Beta Actin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab8226 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

All lanes : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution

Lane 1 : A431 (Human epidermoid carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human epithelial cell line from embryonic kidney) Whole Cell Lysate
Lane 3 : NIH 3T3 (Mouse embryo fibroblast cell line) Whole Cell Lysate
Lane 4 : PC12 (Rat adrenal gland pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (ab175783) 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 5% Milk before being incubated with ab8226 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (ab175783) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

ab8226 staining beta Actin in human gastric epithelial AGS cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde. Permeabilization and blocking was carried out using 5% BSA containing 0.025% Triton X in TBS for 1 hour at 23°C. Primary antibody was used at 5µg/ml for 1 hour at 23°C. An Alexa Fluor 488^® conjugated goat polyclonal to mouse IgG was used as the secondary antibody at a 1/1000 dilution.

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IHC image of ab8226 staining beta 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 ab8226, 0.1μg/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

Immunofluorescence using ab8226 at 5µg/ml incubated for 1 hour on Rat Colon Cancer cells.

Cells were fixed with ice-cold methanol for 5 mins, then for all following steps, permeabilised in TBS-T for 30 mins, blocked with 5% BSA for 30 mins and then washed in TBS-T. Secondary antibody was Alexa Fluor 488 goat anti-mouse IgG at 1/1000 incubated for 1 hour. Cells were counterstained with DAPI. Image at 400X magnification. All incubations were at room temperature.

The beta actin fibres can be seen arrayed around the edge of the cells.

IHC image of ab8226 staining beta Actin in rat 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 ab8226, 0.5μg/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.

Lane 1 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution
Lane 2 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/10000 dilution
Lanes 3-4 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/500 dilution

Lanes 1-2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : HEK293 (Human epithelial cell line from embryonic kidney) cell lysate
Lane 4 : NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 42 kDa


Exposure time: 10 seconds

All lanes : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 4 : HEK293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 5 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-mouse IgG at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 10 seconds

Western blot image using the Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-20%) with the Prism Ultra Protein Ladder (ab116028) 5µl used. We recommend using our ECL substrate kit (ab65623).

Lanes 1-3 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1 µg/ml (5% BSA BLOCK)
Lanes 4-6 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1 µg/ml (5% MILK BLOCK)

Lanes 1 & 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lanes 2 & 5 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lanes 3 & 6 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

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


Exposure time: 8 minutes