All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/ml

Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 5 : Skeletal Muscle (Human) Tissue Lysate - adult normal tissue

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 ab8224 overnight at 4°C. Antibody binding was detected using a goat anti-mouse Alexa Fluor 790 (ab175783) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

IHC image of ab8224 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 ab8224, 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

Overlay histogram showing HeLa cells stained with ab8224 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab8224, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was Mouse IgG3 [MG3-35] (ab18394, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

Performed under reducing conditions.

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


Exposure time: 3 minutes

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% Bovine Serum Albumin before being incubated with ab8224 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406

IHC image of beta actin staining in human colon formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab8224, 3µg/ml overnight at +4°C. A goat anti-mouse HRP-conjugated secondary antibody (ab6789, 1/2000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

The inset negative control image is secondary-only at 1/500 dilution.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) + Xenopus embryo lysate at 20 µg

Secondary
Rabbit Anti-Mouse IgG H&L (HRP) (ab6728)

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa

ab8224 used on Xenopus embryo lysate (20 ug of lysate/lane).

Secondary
Rabbit polyclonal anti-mouse HRP was used as the secondary antibody (ab6728) and developed using the ECL technique.
Performed under reducing conditions.

Predicted band size : 42kD

All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/ml

Lane 1 : Drosophila lysate
Lane 2 : S. pombe lysate
Lane 3 : S. cerevisiae lysate (Actin 1 - please see note)

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

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

Note: although S. cerevisae is not known to express beta Actin, Abcam believes that the band on lane 3 corresponds to Actin 1 (Swissprot ID: P60010, based on sequence similarity).

 

Secondary antibody - rabbit anti-mouse HRP (ab6728)

All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1/1000 dilution

Lane 1 : Fruit fly (Drosophila melanogastor) whole cell lysate - Female
Lane 2 : Fruit fly (Drosophila melanogastor) whole cell lysate -
Male

Lysates/proteins at 100 µg per lane.

Secondary
All lanes : An HRP-conjugated Sheep polyclonal to mouse IgG at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa


Exposure time: 2 minutes


Blocking step: 5% Milk for 1 hour at 20°C.

See Abreview

Immunohistochemistical detection of beta Actin using antibody [mAbcam 8224] - Loading Control on formaldehyde-fixed paraffin-embedded rat cerebellum sections. Antigen retrieval step: heat mediated Citric acid pH6 buffer.  Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary antibody dilution 1/1000 for 2 hours in TBS/BSA/azide. Secondary Antibody: anti Mouse Igs conjugated to biotin (1/200). beta Actin appears to be particularly enriched not only in the glomeruli of the Granule cell layer (indicated by red arrowheads ) but also in Microglia (indicated by green arrowheads); All positive microglia appear to be ramified thus not presumed to be activated.

See Abreview