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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human beta Actin aa 1-100 conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
(Peptide available as
This antibody has been designed for use as a loading control and is ideal for this purpose.
Our Abpromise guarantee covers the use of ab8229 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 5 µg/ml.|
|IHC-P||Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.|
|WB||1/500. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa).Can be blocked with Human beta Actin peptide (ab13772).|
ICC/IF image of ab8229 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab8229, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal donkey serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 donkey anti-goat IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
IHC image of beta Actin staining in Human normal 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 ab8229, 10µg/ml overnight at +4°C. An HRP-conjugated secondary (Ab6885), donkey anti-goat HRP (IgG - H&L; 1/250 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.
The inset negative control image is taken from an identical assay without primary antibody.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
This blot was produced using a 10% 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% Bovine Serum Albumin (lanes 1-4) or 3% milk (lanes 5-8) before being incubated with ab8229 overnight at 4°C. Antibody binding was detected using an anti-Goat antibody conjugated to HRP, and visualised using ECL development solution. A non-specific band at 50-kDa is observed on western blots of some of our batches of ab8229. We have found that milk blocking is more effective than BSA blocking in eliminating this non-specific binding.