The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
1/500 - 1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 60 kDa).
Catalyzes the first step in the oxidation of the side chain of sterol intermediates; the 27-hydroxylation of 5-beta-cholestane-3-alpha,7-alpha,12-alpha-triol. Has also a vitamin D3-25-hydroxylase activity.
All lanes : Anti-CYP27A1 antibody (ab64889) at 1/500 dilution
Lane 1 : extracts from HeLa cells (5-30µg total protein) Lane 2 : extracts from HeLa cells (5-30µg total protein) and 5-10µg of the immunising peptide.
Predicted band size : 60 kDa Observed band size : 60 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - CYP27A1 antibody (ab64889)This image is courtesy of an anonymous Abreview
ab64889 staining CYP27A1 in human colon tissue sections by IHC-P (formaldehyde-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde and blocked with 0.4% Casein in phosphate-buffered saline for 30 minutes at 25°C. Antigen retrieval was by heat mediation in target retrieval solution. Samples were incubated with primary antibody 1/2000 (TBST) for 1 hour at 25°C.
ICC/IF image of ab64889 stained MCF7 cells. The cells were 4% PFA 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 (ab64889, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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.
Bergadà L et al. Role of local bioactivation of vitamin D by CYP27A1 and CYP2R1 in the control of cell growth in normal endometrium and endometrial carcinoma. Lab Invest94:608-22 (2014).
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