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Synthetic peptide conjugated to KLH derived from within residues 350 to the C-terminus of Human Actin.
(Peptide available as ab13771.)
Our Abpromise guarantee covers the use of ab1801 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/1000. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa). Block in 5% BSA. Blocking in milk significantly reduces the signal.|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
IHC image of ab1801 staining Actin in normal 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 ab1801, 5μl/ml 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 negative 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
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 ab1801 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406
Western blot analysis of HeLa cells treated for 12 hours with hesperidin (h) (2.5 μg/ml, 4,01 μM), mangiferin (5 μg/ml, 11.84 μM) (m), and hesperidin (2.5 μg/ml, 4.01 μM) in a presence of mangiferin (5 μg/ml, 11.84 μM) (h+m). Immunoblotting was performed with the following primary antibodies: Bax (ab32503), BCL2 (ab59348), beta actin (ab1801), and caspase 8. After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H+L) or with goat anti-mouse IgG (H+L) HRP-conjugated secondary antibodies and detected using ECL. Densitometry was performed using Image Lab software v. 4.1 (BioRad).
Top panel: Following 12h of treatment of HeLa cells with hesperidin (h), mangiferin (m), and hesperidin in a presence of mangiferin (h+m), the mRNA levels were monitored in real - time PCR experiments. The BAX and BCL2 mRNA levels results from 2 independent experiments (n?=?8) are plotted relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S rRNA levels and expressed as a fold change over the EtOH control. Error bars represent standard derivations.
Bottom panel: Following 12h of treatment of HeLa cells with hesperidin (h), mangiferin (m), and hesperidin in a presence of mangiferin (h+m), the protein levels of Bax and BCL2 were detected with SDS-PAGE and Western Blot and related to beta actin levels.
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