Western Blot Analysis of sFRP1 and ADAM10

Western blot analyses were conducted under reducing conditions as described before [23 (link)]. Specially, 16 μg protein of each TBS-T fraction and 1.2 μg protein of each FA fraction were loaded per well. The primary antibodies and their dilutions used in this study are as follows: anti-sFRP1 (Invitrogen, MA5–38193, 1:1000), anti-ADAM10 (Santa Cruz Biotechnology, SC-28358, 1:1000) and anti-β-actin (Sigma Life Sciences, A2228, 1:5000). The membrane for detecting sFRP1 in FA fraction was restained with Revert 700 total protein stain solution (LI-COR Biosciences) for total protein quantification.

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Macyczko J.R., Wang N., Lu W., Jeevaratnam S., Shue F., Martens Y., Liu C.C., Kanekiyo T., Bu G, & Li Y. (2023). Upregulation of sFRP1 is more profound in female than male 5xFAD mice and positively associated with amyloid pathology. Journal of Alzheimer's disease : JAD, 95(2), 399-405.

Publication 2023

anti-ADAM10 anti-β-actin Revert 700 total protein stain solution

Corresponding Organization : Mayo Clinic in Florida

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Variable analysis

independent variables

Reducing conditions

dependent variables

Protein levels of sFRP1, ADAM10, and β-actin

control variables

Amount of protein loaded per well (16 μg for TBS-T fraction, 1.2 μg for FA fraction)
Primary antibody dilutions (anti-sFRP1 1:1000, anti-ADAM10 1:1000, anti-β-actin 1:5000)

controls

Revert 700 total protein stain solution (LI-COR Biosciences) used to quantify total protein for sFRP1 in FA fraction

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