The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
1/1000 - 1/5000. Detects a band of approximately 75 kDa (predicted molecular weight: 69 kDa).
Use at 2 µg/mg of lysate.
RelevanceRBM14 is a RNA-binding protein. These proteins contribute to gene expression by regulating the form, abundance and stability of both coding and non-coding RNAs. In vertebrates, RNA binding proteins can also influence many processes involved in RNA processing, for example, activity-dependent transcript localization and localized protein synthesis amongst others.
Detection of RBM14 by Western Blot of Immunprecipitate.
ab70636 at 1µg/ml staining RBM14 in HeLa whole cell lysate immunoprecipitated using ab70636 at 3µg/mg lysate (1 mg/IP; 20% of IP loaded/lane).
Detection: Chemiluminescence with exposure time of 30 seconds.
ICC/IF image of ab70636 stained HepG2 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 (ab70636, 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.
IHC image of ab70636 staining in human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with EDTA (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab70636, 10µg/ml, 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.
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.
References for Anti-RBM14 antibody (ab70636)
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