Analysis of proteostasis during aging with western blot of detergent-soluble and insoluble protein fractions

Abstract

Summary Defects in protein quality control are the underlying cause of age-related diseases. The western blot analysis of detergent-soluble and insoluble protein fractions has proven useful in identifying interventions that regulate proteostasis. Here, we describe the protocol for such analyses in Drosophila tissues, mouse skeletal muscle, human organoids, and HEK293 cells. We describe key adaptations of this protocol and provide key information that will help modify this protocol for future studies in other tissues and disease models. For complete details on the use and execution of this protocol, please refer to Rai et al. (2021) and Hunt el al. (2021) .

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy

“…WT or PACSIN1 KO HeLa cells were treated with 20 μM MG132 or 2.5 μg/mL puromycin for 6 h. Detergent soluble-insoluble proteins were fractionated using 1% triton-X 100 with PBS [ 62 ]. After centrifugation, the supernatant was used as the soluble fraction.…”

Integrated genomic and proteomic analyses identify stimulus-dependent molecular changes associated with distinct modes of skeletal muscle atrophy

“…Detergent-soluble protein fractions were extracted by homogenizing tissues in NP40 cell lysis buffer (Invitrogen #FNN0021; 100 μL per ~5 mg of mouse muscle tissue) by using a bullet blender (NextAdvance) with zirconium 0.5-mm beads, and then centrifuging at 15,000 rpm for 10 minutes to pellet insoluble material ( Hunt et al, 2021 ; Rai et al, 2021b ). The supernatant (detergent-soluble protein fraction) was removed and the protein concentration determined by using the Bio-Rad protein assay.…”

Modulation of protease expression by the transcription factor Ptx1/PITX regulates protein quality control during aging

“…Western blots for detergent-soluble and insoluble fractions were done as before. 20 , 32 , 86 In brief, thoraces were dissected from 20 male flies/sample and homogenized in ice-cold PBS with 1% Triton X-100 containing protease and phosphatase inhibitors. Homogenates were centrifuged at 14,000 rpm at 4°C and supernatants collected (Triton X-100 soluble fraction).…”