The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
1/200 - 1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/2000 - 1/10000. Detects a band of approximately 62 kDa (predicted molecular weight: 62 kDa).
Use at 1-4 µg/mg of lysate.
May be required to maintain the proliferative capacity of stem cells and may play an important role in tumorigenesis.
Increased levels in lung tissue in cancer patients.
Belongs to the MMR1/HSR1 GTP-binding protein family. Contains 1 G (guanine nucleotide-binding) domain.
The basic domain (B) allows nucleolar localization in the absence of GTP. The intermediate domain (I) inhibits nucleolar localization by the B domain and is required for exit from the nucleolus. Exit from the nucleolus to the nucleoplasm requires both the I and the acidic (A) domains, and may be triggered by GTP hydrolysis. In contrast to other GTP-binding proteins, this family is characterized by a circular permutation of the GTPase motifs described by a G4-G1-G3 pattern.
Nucleus. Nucleus > nucleolus. Shuttles between the nucleus and nucleolus.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skin carcinoma tissue labelling Nucleostemin with ab70346 at 1/1000 (0.2µg/ml). Detection: DAB.
Western blot - Nucleostemin antibody (ab70346)
All lanes : Anti-Nucleostemin antibody (ab70346) at 0.04 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg Lane 5 : mouse 3T3 whole cell lysate at 50 µg
Predicted band size : 62 kDa Observed band size : 62 kDa
Detection of Nucleostemin by Western Blot of Immunprecipitate.
All lanes: ab70346 at 1µg/ml staining Nucleostemin in HeLa whole cell lysates immunoprecipitated at 3µg/mg lysate (1 mg/IP; 20% of IP loaded/lane) using the following antibodies:
Lane 1: an irrelevant antibody.
Lane 2: ab70345
Lane 3: ab70346.
Detection: Chemiluminescence with exposure time of 3 seconds
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nucleostemin antibody (ab70346)This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom
Immunohistochemistical detection of Nucleostemin with ab70346 on formaldehyde-fixed paraffin-embedded human colon sections. Antigen retrieval step: heat mediated in citric acid pH6 buffer. Blocking step: 1% BSA for 10 mins @ rt°C. Primary antibody ab70346 incubated at 1/50 for 16 hours in TBS/BSA/azide. Secondary antibody: anti-rabbit IgG conjugated to biotin (1/200). The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. Stem cell markers have been observed in cycling columnar cells at the crypt base (Nature 449, 1003-1007; 2007). The cells (nuclei of the simple columnar epithelium of the upper mucosa) immunoreactive for Nucleostemin in this image (green arrowheads) anatomically correspond to the same cells expected to be Lgr5 positivite. A red arrowhead indicates nucleolar positivity in one of many cell nuclei in the Lamina propria of the mucosa (this area is not known for stem cell posit
ICC/IF image of ab70346 stained HeLa cells. The cells were 4% formaldehyde 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 (ab70346, 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.