Determination of Cell Surface Protein Levels

For determination of relative cell surface protein levels, cells were treated with Sulfo-NHS-LC-Biotin (Pierce; 0.25 mg/ml PBS supplemented with 1 mM MgCl₂ and 0.1 mM CaCl₂), quenched with 100 mM glycine in DMEM and lysed in lysis buffer supplemented with Complete Proteinase Inhibitor Cocktail Tablets (Roche). Biotinylated proteins were immunoprecipitated using NeutrAvidin beads (Thermo Scientific) and analyzed by Western blotting. For detailed information see (Braune et al., 2014 (link); Preuße et al., 2015 (link)).

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Tveriakhina L., Schuster-Gossler K., Jarrett S.M., Andrawes M.B., Rohrbach M., Blacklow S.C, & Gossler A. (2018). The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro. eLife, 7, e40045.

Publication 2018

Complete Proteinase Inhibitor Cocktail Tablets NeutrAvidin beads

Buffer Cell surface protein Cells Glycine Mgcl2 Neutravidin Proteinase inhibitor Proteins Sulfo nhs lc biotin

Corresponding Organization :

Other organizations : Medizinische Hochschule Hannover, Harvard University, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Sulfo-NHS-LC-Biotin (Pierce; 0.25 mg/ml PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2)

dependent variables

Relative cell surface protein levels

control variables

Glycine (100 mM in DMEM) used to quench the biotinylation reaction
Lysis buffer supplemented with Complete Proteinase Inhibitor Cocktail Tablets (Roche) used to lyse the cells

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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