Anti-NMNAT2 antibody [2E4]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NMNAT2 antibody [2E4] (AB56980)

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.

This image was generated using the ascites version of the product.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NMNAT2 antibody [2E4] (AB56980)

IHC image of ab56980 staining in human breast cancer formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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.

This image was generated using the ascites version of the product.

• Flow Cyt

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Flow Cytometry - Anti-NMNAT2 antibody [2E4] (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 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.
This image was generated using the ascites version of the product.

• WB

AbReview33950****

Western blot - Anti-NMNAT2 antibody [2E4] (AB56980)

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]

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-NMNAT2 antibody [2E4] (ab56980) at 2 µg/mL

All lanes:

mouse brain from newborn pup at 40 µg

Secondary

All lanes:

Goat Anti-Mouse IgG (H+L)-HRP Conjugate at 1/3000 dilution

Predicted band size: 34 kDa

false

This image is courtesy of an abreview submitted by Jon Gilley, The Babraham Institute, United Kingdom

• sELISA

Supplier Data

Sandwich ELISA - Anti-NMNAT2 antibody [2E4] (AB56980)

Detection limit for recombinant GST tagged NMNAT2 is 0.03 ng/ml as a capture antibody.

• WB

Supplier Data

Western blot - Anti-NMNAT2 antibody [2E4] (AB56980)

Western blot against tagged recombinant protein immunogen using ab56980 NMNAT2 antibody at 1ug/ml. Predicted band size of immunogen is 37 kDa

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-NMNAT2 antibody [2E4] (ab56980)

Predicted band size: 34 kDa

false