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Synthetic peptide derived from residues 600 to the C-terminus of Human NUMB.
Our Abpromise guarantee covers the use of ab14140 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 8 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
|WB||Use a concentration of 0.5 - 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 70 kDa).Can be blocked with Human NUMB peptide (ab14141).|
|IP||Use at an assay dependent concentration.|
Image courtesy of Human Protein Atlas
ab14140 staining NUMB in Human adrenal gland. The paraffin embedded human tissue was incubated with ab14140 (1/250 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab14140 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.
Further results for this antibody can be found at www.proteinatlas.org
ab14140 detecting NUMB protein in dissociated neural precursor cells cultured from embryonic day 13 mouse neocortex in the presence of bFGF, EGF and LIF. NUMB immunoreactivity (green) was found in the cytosol of neural precursor cells. This localisation is in general agreement with published studies (e.g. Chen et al., EJN, 2005), where similar labelling is observed in the cytosol of cells of the grey matter of adult mouse spinal cord. Immunocytochemistry: All steps were performed in PBS. Cells were fixed in 4% PFA for 15min, permeabilised with 0.1% TX100 for 10min and blocked with 5% BSA, 0.1% TX100 for 45min. ab14140 was incubated at 8µg/ml for 12h at 4°C in 5% BSA, 0.1% TX100. Cultures were washed (3x) of primary antibody solution. Goat anti-rabbit AlexaFluor 488 was used as secondary antibody (1/400) in 5% BSA, 0.1% TX100 for 1h at RT. To-pro-3 was used as a nuclear counterstain (blue). Treated cultures were mounted on glass coverslips with Mowiol.
ab14140 at 1/100 staining rat brain tissue sections by IHC-Fr. The tissue was paraformaldehyde fixed and blocked with 5% serum prior to incubation with the antibody for 24 hours. An Alexa-Fluor ® 488 conjugated goat anti-rabbit antibody was used as the secondary. NUMB staining is shown in green.
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 ab14140 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
Denaturing of the protein sample was imperative to the success of this WB (denatured at 95°C for 15min). An additional, higher band (around 80kDa) is seen if not denatured. Original concentration tried was 1ug/ml but we found this high given the circumstances and have since used 0.5ug/ml. Theoretically, even less could be used.
ab14140 staining NUMB in Zebrafish embryo nervous system by Immunohistochemistry (Whole mount). Samples were incubated with primary antibody (1/500 in BSA blocking buffer) for 48 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-rabbit polyclonal (1/500) was used as the secondary antibody.
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