The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
Use a concentration of 1 - 5 µg/ml. This antibody has only been tested in WB against the recombinant fragment used as immunogen. We have no data on the detection of endogenous protein.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Catalyzes the formation of NAD(+) from nicotinamide mononucleotide (NMN) and ATP. Can also use the deamidated form; nicotinic acid mononucleotide (NaMN) as substrate but with a lower efficiency. Cannnot use triazofurin monophosphate (TrMP) as substrate. Also catalyzes the reverse reaction, i.e. the pyrophosphorolytic cleavage of NAD(+). For the pyrophosphorolytic activity prefers NAD(+), NADH and NAAD as substrates and degrades nicotinic acid adenine dinucleotide phosphate (NHD) less effectively. Fails to cleave phosphorylated dinucleotides NADP(+), NADPH and NAADP(+).
Highly expressed in brain, in particular in cerebrum, cerebellum, occipital lobe, frontal lobe, temporal lobe and putamen. Also found in the heart, skeletal muscle, pancreas and islets of Langerhans.
Cofactor biosynthesis; NAD(+) biosynthesis; NAD(+) from nicotinamide D-ribonucleotide: step 1/1.
Belongs to the eukaryotic NMN adenylyltransferase family.
ICC/IF image of ab56980 stained SHSY5Y cells. The cells were 100% methanol fixed (5 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 (ab56980, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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 ab56980 staining in human breast cancer 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 sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab56980, 5µg/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.
Flow Cytometry - NMNAT2 antibody (ab56980)
Overlay histogram showing SH-SY5Y cells stained with ab56980 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56980, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
Western blot - Anti-NMNAT2 antibody (ab56980)This image is courtesy of an abreview submitted by Jon Gilley, The Babraham Institute, United Kingdom
Anti-NMNAT2 antibody (ab56980) at 2 µg/ml + mouse brain from newborn pup at 40 µg
Secondary Goat Anti-Mouse IgG (H+L)-HRP Conjugate at 1/3000 dilution
This image is courtesy of an abreview submitted by Jon Gilley, The Babraham Institute, United Kingdom
Nmnat2 is a low abundance protein and is difficult to detect. Although multiple non-specific bands are detected by this antibody, we find it is the best commercial antibody for detecting endogenous Nmnat2 as the band is found approximately mid-way between the 25 and 37 kDa markers (at ~32 kDa) and can relatively easily be distinguished from nearby non-specific bands (N.B. a non-specific band at ~37 kDa is also detected in mouse brain extracts from adult mice). Importantly, we do not use this antibody in any immunostaining techniques due to its cross-reactivity with multiple other proteins.
The size of the specific band corresponding to endogenous Nmnat2 (32 kDa) was determined based on its migration relative to that of FLAG-tagged Nmnat2 in tranfected HEK cells (which migrates slightly slower at ~34 kDa) and loss of the endogenous Nmnat2 band in transected axons in primary neuronal cultures and in gene trap mouse brain extracts (Gilley and Coleman, PLoS Biology, 2010 [PMID: 20126265]