Application
Western blot
Sample
Mouse Tissue lysate - whole (brain from newborn pup)
Loading amount
40 µg
Specification
brain from newborn pup
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Other product details
Concentration
2 µg/ml
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBS, 0.05% Tween, 5% milk
Secondary antibody
Name
Non-Abcam antibody was used: Goat Anti-Mouse IgG (H+L)-HRP Conjugate #172-1011
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/3000
Detection
Detection method
ECL+
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: ~32 kDa Non-specific: multiple (see blot) kDa
Positive control
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] Hicks et al., PLoS One, 2012 [PMID: 23082226]).
Additional data
Additional Notes
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.
Abcam response
Many thanks for your feedback in mouse WB with ab56980 demonstrating reactivity with mouse NMNAT2 protein.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
DR. Jon Gilley
Verified customer
提交于 Jan 09 2013