Application
Western blot
Sample
Mouse Tissue lysate - other (brain)
Gel Running Conditions
Non-reduced Denaturing (10% acrylamide)
Loading amount
25 µg
Treatment
25uM DAPT
Specification
brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% BSA in TBS-0.05% Tween20
Dilution
1/1000
Secondary antibody
Dilution
1/5000
Name
Non-Abcam antibody was used: Donkey anti-rabbit IRDye 800 CW
Host species: Donkey
Clonality: Polyclonal
Conjugation: IRDye® 800CW
Host species: Donkey
Clonality: Polyclonal
Conjugation: IRDye® 800CW
Detection
Detection method
Odyssey-IR fluorescence scanner
Negative control
Gamma-secretase inhibitor addition: DAPT 25 uM
DAPT References: Zou et al. Clin Exp Med. 2013. 13:271-8 Floyd et al. Neuro Oncol. 2012. 14: 1215–1226.
Exposure
5 minute(s) and 0 second(s)
Bands
Specific: 300, 110-125, 95 (NI kDa Non-specific: N/A kDa
Positive control
Gamma-secretase activity assay with endogenous substrate in mouse brain cortex samples incubated for 2 hrs to accumulate the NICD product and membrane/soluble fractions isolated to detect the specific band (protocol described in Torres et al. Mol Neurodegener. 2012. 7:59)
M: Membrane fraction
S: Soluble fraction
Additional data
Additional Notes
The antibody detects the full Notch and its different short forms (after fractionation by Furin, ADAM and g-secretase). It is possible to detect NICD after its accumulation by g-secretase assay (Torres et al. Mol Neurodegener. 2012. 7:59) and the resulting band is identified because it disappears with DAPT incubation.
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.
Ms. Victoria Lladó
Verified customer
投稿 Oct 16 2015