The section was then counterstained with haematoxylin and mounted with DPX. DAB was used as the chromogen ( ab103723), diluted 1/100 and incubated for 10min at room temperature. Streptavidin HRP was used, ab7403 at a 1/10000 dilution. A goat anti-rabbit biotinylated secondary antibody (ab6720, 1/1000 dilution) was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was then incubated with ab8227, 1/1000 dilution, for 15 mins at room temperature. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. IHC image of beta actin staining in a section of formalin-fixed paraffin-embedded normal human colon*. Antibody binding was detected using Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) secondary antibody at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx. The membrane was then blocked for an hour using 5% Milk before being incubated with ab8227 overnight at 4☌. The gel was run at 200V for 50 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. Observed band size: 42 kDa why is the actual band size different from the predicted? Lane 4 : PC-12 (rat adrenal gland pheochromocytoma cell line) Whole Cell LysateĪll lanes : Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) ( ab175781) at 1/10000 dilution Lane 3 : NIH/3T3 (mouse embryo fibroblast cell line) Whole Cell Lysate Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) Whole Cell Lysate Lane 1 : A431 (human epidermoid carcinoma cell line) Whole Cell Lysate All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution
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