Western Blot Analysis of Alzheimer's Markers

Immunoblots was performed as described previously [16 (link)]. Briefly, for Western blot analysis, samples (30 μg protein) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and were then transferred to polyvinylidene difluoride (PVDF) membranes. The primary antibodies used were as follows: mouse anti-GFAP antibody (GFAP, Millipore, MAB360, 2041104), mouse anti-APP antibody (Millipore, MAB348, 25030193), rabbit anti-CTF antibody (Millipore, AB5352, 22051154), rabbit anti-neprilysin (NEP) antibody (Millipore, AB5458, LV1423485), rabbit anti-IDE antibody (Millipore, AB9210, 2769024), mouse anti-β-actin antibody (Millipore, MAB, LV1460528) and goat anti-Iba-1 antibody (Abcam, ab5076, GR268568-3). The secondary antibodies were anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP; Jackson ImmunoResearch, 711-035-152) and anti-mouse IgG antibody conjugated with HRP (Jackson ImmunoResearch, 715-035-151). The immune complexes were detected using enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA). The images were obtained and quantified using a LAS-3000 Image Analyzer (Fujifilm, Tokyo, Japan).

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Tzeng T.T., Chen C.C., Chen C.C., Tsay H.J., Lee L.Y., Chen W.P., Shen C.C, & Shiao Y.J. (2018). The Cyanthin Diterpenoid and Sesterterpene Constituents of Hericium erinaceus Mycelium Ameliorate Alzheimer’s Disease-Related Pathologies in APP/PS1 Transgenic Mice. International Journal of Molecular Sciences, 19(2), 598.

Publication 2018

MAB360 MAB348 AB9210 ab5076 enhanced chemiluminescence reagents LAS-3000 Image Analyzer

Actin Anti antibody Anti gfap antibody Anti igg Antibodies Antibody Chemiluminescence Gfap Goat Immune complexes Immunoblots Membranes Mouse Neprilysin Polyvinylidene difluoride Protein Rabbit Sds polyacrylamide gel electrophoresis Western blot analysis

Corresponding Organization :

Other organizations : National Yang Ming Chiao Tung University, Chang Gung University of Science and Technology, Grape King (Taiwan), National Research Institute of Chinese Medicine, Ministry of Health and Welfare

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of GFAP
Levels of APP
Levels of CTF
Levels of NEP
Levels of IDE
Levels of β-actin
Levels of Iba-1

control variables

Amount of protein loaded (30 μg)
Use of SDS-PAGE for protein separation
Transfer of proteins to PVDF membranes
Primary antibodies used (mouse anti-GFAP, mouse anti-APP, rabbit anti-CTF, rabbit anti-NEP, rabbit anti-IDE, mouse anti-β-actin, goat anti-Iba-1)
Secondary antibodies used (anti-rabbit IgG-HRP, anti-mouse IgG-HRP)
Detection method (enhanced chemiluminescence)
Imaging and quantification (LAS-3000 Image Analyzer)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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