Immobilization and Validation of Tau-13 Antibody

The tau-13 antibody was immobilized as previously described^{52 (link)}. Briefly, the antibody was covalently linked to Protein G magnetic beads (Thermo Fisher Scientific; 200 µg of antibody per 1 mL of bead slurry) using dimethyl pimelimidate (Thermo Fisher Scientific) as the crosslinker. Prior to use, beads were pre-treated with IP buffers A and B to wash off any antibody not covalently linked to beads. To monitor any shedding of antibody from the beads, the beads were treated in elution buffer (100 mM glycine-HCl (pH 2.8) and 1% (w/v) n-Octyl β-D-thioglucopyranoside (OTG)). The eluate was collected and analyzed by WB using HRP-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA).

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Liu P., Smith B.R., Montonye M.L., Kemper L.J., Leinonen-Wright K., Nelson K.M., Higgins L., Guerrero C.R., Markowski T.W., Zhao X., Petersen A.J., Knopman D.S., Petersen R.C, & Ashe K.H. (2020). A soluble truncated tau species related to cognitive dysfunction is elevated in the brain of cognitively impaired human individuals. Scientific Reports, 10, 3869.

Publication 2020

Protein G magnetic beads dimethyl pimelimidate HRP-conjugated goat-anti-mouse IgG

Anti igg Antibody Buffer Dimethyl pimelimidate Glycine Goat Mouse Protein g

Corresponding Organization :

Other organizations : University of Minnesota, Mayo Clinic, WinnMed

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Variable analysis

independent variables

Immobilization of tau-13 antibody on Protein G magnetic beads using dimethyl pimelimidate as the crosslinker

dependent variables

Shedding of antibody from the beads, as measured by Western blot analysis using HRP-conjugated goat-anti-mouse IgG

control variables

Pre-treatment of beads with IP buffers A and B to wash off any antibody not covalently linked to beads
Elution of beads in 100 mM glycine-HCl (pH 2.8) and 1% (w/v) n-Octyl β-D-thioglucopyranoside (OTG) buffer

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

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