Anti-beta Actin antibody [mAbcam 8224] - Loading Control

• WB

Lab

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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.

All lanes:

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/mL

Lane 1:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Lane 3:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg

Lane 4:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 20 µg

Lane 5:

Skeletal Muscle (Human) Tissue Lysate - adult normal tissue at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-alexa-fluor-790-ab175783'>ab175783</a>) at 1/10000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

ab8224 staining beta Actin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab8224 at 1µg/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 magenta). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling beta actin with ab8224 at a concentration of 0.01µg/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. ab8224 anti-beta Actin antibody [mAbcam 8224] 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

AbReview18268****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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.

This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom

• WB

Lab

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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

All lanes:

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 3:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/50000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa

false

Exposure time: 3min

• WB

AbReview81844****

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

Western blot analysis using ab8224 at 1 : 1000 on Mouse CT26 cells. Blocking agent and dilution buffer was 5% milk in TBST

All lanes:

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1/10000 dilution

All lanes:

Mouse CT26 cells at 40 µg

Secondary

All lanes:

Anti-mouse IgG, HRP-linked Antibody at 1/10000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa

false

This image is courtesy of an Abreview submitted by Joel Ohana

• WB

AbReview17900****

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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

All lanes:

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1/1000 dilution

Lane 1:

Fruit fly (Drosophila melanogastor) whole cell lysate - Female at 100 µg

Lane 2:

Fruit fly (Drosophila melanogastor) whole cell lysate - Male at 100 µg

Secondary

All lanes:

An HRP-conjugated Sheep polyclonal to mouse IgG at 1/10000 dilution

Predicted band size: 41 kDa

true

Exposure time: 2min

This image is courtesy of an anonymous Abreview

• WB

Project390****

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

Note : although S. cerevisiae 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:

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/mL

Lane 1:

Drosophila lysate at 20 µg

Lane 2:

S. pombe lysate at 20 µg

Lane 3:

S. cerevisiae lysate (Actin 1 - please see note) at 20 µg

Secondary

All lanes:

Western blot - Rabbit Anti-Mouse IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/rabbit-mouse-igg-h-l-hrp-ab6728'>ab6728</a>) at 1/5000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa

true

• WB

Project

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (AB8224)

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

SecondaryRabbit polyclonal anti-mouse HRP was used as the secondary antibody (ab6728) and developed using the ECL technique.
Predicted band size : 42kD

All lanes:

Western blot - Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224)

All lanes:

Xenopus embryo lysate at 20 µg

Secondary

All lanes:

Western blot - Rabbit Anti-Mouse IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/rabbit-mouse-igg-h-l-hrp-ab6728'>ab6728</a>)

Predicted band size: 41 kDa

true