Western Blot Analysis of Brain apoE

PBS-soluble brain lysates from the sequential homogenization step were analyzed for total protein concentration with a micro BCA kit (Thermo Scientific). Thirty microgram of proteins from each sample were loaded onto a NU-PAGE 4–12% Bis-Tris 15 well gel (Thermo Fisher Scientific # NP0336BOX) and the gel was run at 150 V for 1.5 h. The proteins were subsequently dry-transferred onto a PVDF membrane using the iblot2 system (Life Technologies) and blocked with 5% milk in TBS-Tween (0.05%). The membrane was incubated with anti-apoE antibody HJ15.7 [34 (link)] (or HJ15.3) and anti-β-tubulin antibodies to probe for apoE and a loading control, respectively. Donkey-anti-mouse IgG-HRP was used as secondary antibody (Santa Cruz Biotechnology # sc-2096). All blots were developed for ~ 10 s using an enhanced chemiluminescence (ECL) Ultra kit (Lumigen TMA-6) and imaged on the SynGene Imager (BioRad) at the appropriate exposure.

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Huynh T.P., Wang C., Tran A.C., Tabor G.T., Mahan T.E., Francis C.M., Finn M.B., Spellman R., Manis M., Tanzi R.E., Ulrich J.D, & Holtzman D.M. (2019). Lack of hepatic apoE does not influence early Aβ deposition: observations from a new APOE knock-in model. Molecular Neurodegeneration, 14, 37.

Publication 2019

micro BCA kit iblot2 system Donkey-anti-mouse IgG-HRP

Anti antibodies Anti antibody Anti igg Antibody Apoe Bis tris Brain Chemiluminescence Donkey Membrane Milk Mouse Proteins Pvdf Tubulin Tween

Corresponding Organization :

Other organizations : Hope Center for Neurological Disorders, Harvard University, Massachusetts General Hospital

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Variable analysis

independent variables

Sequential homogenization step to prepare PBS-soluble brain lysates

dependent variables

Total protein concentration measured by micro BCA kit
Apolipoprotein E (apoE) protein levels measured by anti-apoE antibody HJ15.7 or HJ15.3
β-tubulin protein levels as a loading control

control variables

Gel type: NU-PAGE 4–12% Bis-Tris 15 well gel
Gel run conditions: 150 V for 1.5 h
Protein transfer: Dry transfer using iblot2 system
Blocking solution: 5% milk in TBS-Tween (0.05%)
Secondary antibody: Donkey-anti-mouse IgG-HRP
Imaging: Enhanced chemiluminescence (ECL) Ultra kit and SynGene Imager

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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