Covalent Protein Conjugation to Beads

A1WT, A1R1450E and antibodies were pre-coupled covalently with maleimide-PEG3500-NHS (MW ~3500; JenKem, TX). As previously described (Ju et al., 2013a (link), 2015a (link)), the modified proteins were then mixed with streptavidin (SA)-maleimide (Sigma-Aldrich, St. Louis, MO) in carbonate/bicarbonate buffer (pH 8.5) and together linked to silanized borosilicate beads (Thermo Fisher Scientific, Waltham, MA) in phosphate buffer (pH 6.8). Site densities of ligands on beads were measured using the previously described flow cytometry method (Ju et al., 2015a (link)).

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Ju L., Chen Y., Xue L., Du X, & Zhu C. (2016). Cooperative unfolding of distinctive mechanoreceptor domains transduces force into signals. eLife, 5, e15447.

Publication 2016

streptavidin (SA)-maleimide

Antibodies Bicarbonate Buffer Carbonate Flow cytometry Ligands Maleimide Phosphate Proteins Streptavidin

Corresponding Organization :

Other organizations : University of Sydney, The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, The Heart Research Institute, Pennsylvania State University, University of Illinois Chicago

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Variable analysis

independent variables

A1R1450E
Antibodies

dependent variables

Site densities of ligands on beads

control variables

Maleimide-PEG3500-NHS (MW ~3500)
Streptavidin (SA)-maleimide
Carbonate/bicarbonate buffer (pH 8.5)
Phosphate buffer (pH 6.8)
Silanized borosilicate beads

controls

The previously described flow cytometry method (Ju et al., 2015a) was used to measure the site densities of ligands on beads, which serves as a positive control.

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