Protein Expression Analysis in Aging Mouse Brains

2-, 4- and 6-month EFAD mice were anesthetized with sodium pentobarbital (50 mg/kg), transcardially perfused with ice-cold PBS, brains removed and dissected into cortex and hippocampus, snap-frozen in liquid nitrogen and stored at -80°C, as previous described [133 (link)]. Dissected brains were homogenized in lysis buffer [90 (link),132 (link)] (50 mM Tris-HCl, 150 mM NaCl, pH7.4, 1% Triton X-100, 1x protease inhibitor cocktail) and 40 μg of total protein (BCA protein assay kit; Pierce, Rockford, IL) was separated on 4–12% gradient Bis-Tris gels (Invitrogen) under reducing conditions, and transferred to PVDF membranes [47 (link)]. The following primary antibodies were used: rabbit anti-PSD95 (1:3000, Abcam), mouse anti-synaptophysin (1:2000, Abcam), mouse/rabbit anti-β-actin (1:2000; Abcam), rabbit anti-drebrin antibody (1:1000; Abcam), rabbit anti-NMDAR1/anti-NMDAR2B (1:1000; Millipore), anti-NMDAR2A (1:500; Millipore), mouse anti-apoE (1:600; Santa Cruz), rabbit anti-BDNF (1:200; Santa Cruz), rabbit anti-p-CaMK-II (1:1000; Santa Cruz) and rabbit anti-p-CREB (1:1000; Cell Signaling) [90 (link),132 (link)]. HRP-conjugated secondary antibodies, enhanced chemiluminescence (Amersham, Piscataway, NJ) and Image J software were used to quantify densities of the immunoreactive bands relative to β-actin.

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Liu D.S., Pan X.D., Zhang J., Shen H., Collins N.C., Cole A.M., Koster K.P., Ben Aissa M., Dai X.M., Zhou M., Tai L.M., Zhu Y.G., LaDu M.J, & Chen X.C. (2015). APOE4 enhances age-dependent decline in cognitive function by down-regulating an NMDA receptor pathway in EFAD-Tg mice. Molecular Neurodegeneration, 10, 7.

Publication 2015

4–12% gradient Bis-Tris gels rabbit anti-PSD95 mouse anti-synaptophysin rabbit anti-BDNF rabbit anti-p-CaMK-II rabbit anti-p-CREB enhanced chemiluminescence

Actin Anti antibody Anti synaptophysin Antibodies Apoe Assay Bis tris Brains Buffer Camk Chemiluminescence Cold Cortex Frozen Gels Hippocampus Membranes Mouse Nacl Nitrogen Nmdar1 Nmdar2a Nmdar2b Protease inhibitor Protein Pvdf Rabbit Sodium pentobarbital Tris Triton x 100

Corresponding Organization :

Other organizations : Union Hospital, Fujian Medical University, University of Illinois at Chicago

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Age of EFAD mice (2-, 4- and 6-month)

dependent variables

Levels of the following proteins in the cortex and hippocampus of EFAD mice:
PSD95
Synaptophysin
β-actin
Drebrin
NMDAR1
NMDAR2B
NMDAR2A
P-CaMK-II
P-CREB

control variables

Anesthesia with sodium pentobarbital (50 mg/kg)
Transcardial perfusion with ice-cold PBS
Dissection of brains into cortex and hippocampus
Snap-freezing of dissected brains in liquid nitrogen and storage at -80°C
Homogenization of dissected brains in lysis buffer
Quantification of total protein using BCA protein assay kit
Separation of 40 μg of total protein on 4–12% gradient Bis-Tris gels under reducing conditions
Transfer of proteins to PVDF membranes
Use of specific primary and secondary antibodies for Western blot analysis
Quantification of immunoreactive band densities relative to β-actin using Image J software

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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