Quantifying Total Cellular Protein via TIRFM

For detection of total cellular protein (all immunofluorescence experiments), after treatments, as indicated, cells were fixed with 4% paraformaldehyde for 30 min, followed by quenching of fixative in 100 mM glycine, cell permeabilization in 0.1% Triton X-100 (all solutions made in PBS), and then blocking in 3% BSA (Thermo Fisher Scientific). Subsequently, cells were stained with primary and secondary antibodies as indicated and retained within an aqueous medium for imaging by TIRFM.

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Rahmani S., Ahmed H., Ibazebo O., Fussner-Dupas E., Wakarchuk W.W, & Antonescu C.N. (2023). O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits. The Journal of Biological Chemistry, 299(3), 102963.

Publication 2023

BSA

After treatments Antibodies Cells Fixative Glycine Immunofluorescence Paraformaldehyde Protein Triton x 100

Corresponding Organization :

Other organizations : Toronto Metropolitan University, University of Alberta

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Variable analysis

independent variables

Treatments (not explicitly mentioned)

dependent variables

Total cellular protein

control variables

Fixation with 4% paraformaldehyde for 30 min
Quenching of fixative in 100 mM glycine
Cell permeabilization in 0.1% Triton X-100 (all solutions made in PBS)
Blocking in 3% BSA (Thermo Fisher Scientific)
Staining with primary and secondary antibodies as indicated
Imaging by TIRFM

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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