Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation
Transcrição
The degradation of misfolded proteins is crucial for maintaining protein homeostasis in cells.
To examine the degradation of misfolded proteins, take a culture plate containing transfected cells producing GFP-labeled, misfolding-prone proteins. Incubate the plate to facilitate the expression of the transfected protein until aggregates accumulate inside the cells.
Now, treat these cells with cycloheximide – a protein translation inhibitor – for different time intervals, and snap-freeze the cells. Cycloheximide inhibits new protein synthesis; this helps to study the time-dependent degradation of the pre-formed misfolded proteins.
Discard the spent medium, and treat the cells with lysis buffer containing a weak non-ionic detergent. Upon lysis, the detergent molecules solubilize the misfolded protein's monomers and oligomers, while the aggregates remain intact.
Centrifuge the contents to pelletize the aggregates, and discard the supernatant. Dispense the pellet into a sodium dodecyl sulfate, or SDS, buffer, and heat the sample. This treatment denatures the disordered aggregates, while the SDS-insoluble aggregates remain intact.
Load all the samples corresponding to different time intervals of cycloheximide treatment onto the SDS-PAGE gel. Due to their larger size, the SDS-insoluble aggregates remain intact in the well, while the monomers and SDS-soluble proteins migrate in the gel and form distinct bands.
Blot the samples and visualize using anti-GFP-antibodies. After longer cycloheximide treatment, the decreased intensity of the SDS-soluble proteins bands confirms misfolded protein degradation over time.
