Microscopy-based GSH bead protein-protein interaction assay
Xuefeng Ren, Minghao Chen
Published: 2023-10-17 DOI: 10.17504/protocols.io.4r3l27xdxg1y/v1
Abstract
Microscopy-based GSH bead protein-protein interaction assay
Steps
1.
Wash pre-blocked glutathione sepharose beads (GE Healthcare) with the reaction buffer (25 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 1 mM TCEP) three times.
2.
Make a mixture of 1 μM purified GST tagged protein and 500 nM purified fluorescent protein in total 70 μl
3.
Incubate at Room temperature for 0h 30m 0s, samples were mixed with additional 100 μL reaction buffer.
4.
Transferred to the observation chamber for imaging.
5.
Acquire images on a Nikon A1 confocal microscope with a Nikon Plan APO VC 20x/0.75 NA UV Microscope Objective.
