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

All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224)

Lane 1 : Drosophila lysate at 20 µg
Lane 2 : S. pombe lysate
Lane 3 : S. cerevisiae lysate (Actin 1 - please see note)

Secondary
Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6728) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted 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).

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

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

Secondary
Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6728)
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 42 kDa

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

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
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.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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.

Carl Hobbs, King`s College London, United Kingdom

Immunocytochemistry/ Immunofluorescence-beta Actin antibody [mAbcam 8224] - Loading Control(ab8224)

ICC/IF image of ab8224 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8224, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Flow Cytometry-Anti-beta Actin antibody [mAbcam 8224] - Loading Control(ab8224)

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 DyLight® 488 goat anti-mouse 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.