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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human beta Actin.
(Peptide available as ab13772.)
This clone works well as a loading control for Xenopus, Drosophila and S.pombe. We recommend using ab8224 instead of ab8226 for these species.
Note: although S. cerevisae is not known to express beta Actin, Abcam believes a band at similar MW to beta Actin detected in this species corresponds to Actin 1 (Swissprot ID: P60010, based on sequence similarity) - see WB image.
Our Abpromise guarantee covers the use of ab8224 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 1µg for 106 cells. ab18392-Mouse monoclonal IgG3, is suitable for use as an isotype control with this antibody.|
|IHC-P||1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa).Can be blocked with Human beta Actin peptide (ab13772). This antibody has been designed for use as a loading control and is ideal for this purpose. Block membrane for 1 hr in 5%BSA. Incubate antibody in TBST for one hour or more.|
|ICC/IF||Use a concentration of 1 µg/ml.|
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
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.
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
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).
ab8224 used on Xenopus embryo lysate (20 ug of lysate/lane).
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
This image is courtesy of an anonymous AbreviewBlocking step: 5% Milk for 1 hour at 20°C.
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.
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.
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