| Isolation and purification of protein complex is a key and universal method to understand their composition,structure,functional properties.For a certain protein complex,specific modification and development still need to be done due to their unimaginable diversity.For example,RIP3,who tends to form big protein complx in necroptosis,would easily go into the precipitate during high speed centrifugation in normal immuno-precipitation experiments,which leads part of information lost in this process.So how to isolate and purify RIP3 protein complex with high molecular mass is a tricky issue to be solved.In this study,we were the first to use high resolution flow cytometer MoFloAstrios EQ(Beckman Coulter)to analyze and purify fluorescence-labeled RIP3 complex.After feasibility assessment,we labeled RIP3 and its mutants with mScarlet fluorescent protein and over-expressed them to form large aggregates in 293T cell line.Then we made cell lysate and performed differential centrifugation to get a crude RIP3 complex enriched fraction.We passed this fraction through fluorescence-assisted flow cytometery and use100/200/300nm standard letax beads to set proper gate.We sorted mScarlet-positive particles according to their Side scatter(SSC)and mScarlet fluorescence intensity.After concentrating the collected fraction via ultra centrifugation,we performed Sliver Staining to measure the protein yield.And we compared the protein composition between the pre-sorted and sorted fraction with western-Blot,Sliver Staining and DDS-MS.Our data shows the fluorescence-assisted sorting method could purify certain protein complex from 20%to 80%by removing the background proteins,and reach peptites abundance 2-3 fold enrichment.Although a lot of details still need to be discussed and modifications to be done,we have confidence to say that fluorescence-assisted protein complex sorting would give us novel insights and information in the future protein complex studies. |
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