Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells

Domanski, Michal; LaCava, John

doi:10.21769/bioprotoc.2238

Cited by 5 publications

(10 citation statements)

References 16 publications

(25 reference statements)

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“…As examples, pre‐electrophoresis has been used to equilibrate the buffer's ions for reducing the conductivity gradient within the gel . Also, raising the gel temperature before sample loading and separation to maintain the denatured state of DNA samples and to remove free ions that could affect the separation process has been pursued . In addition, a pre‐electrophoresis technique has been used for removing templating agents such as SDS micelles, DNA, and xanthan from nano‐templated polyacrylamide gels …”

Section: Introductionmentioning

confidence: 99%

Influence of pre‐electrophoresis on protein separations in polyacrylamide gels

2020

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Polyacrylamide gel electrophoresis (PAGE) is a widely used technique in protein separations. In this study, the effects of pre‐electrophoresis (i.e., exposing polyacrylamide gels to an electrical field prior to loading samples) on the separation of a mixture of 10 standard proteins was investigated. Three different gel concentrations (based on amounts of acrylamide and bis‐acrylamide, always at a 37.5:1 ratio) relative to the volume of the gel solution were tested: 6%, 9%, and 12%. For each concentration, multiple slab gels were subjected to pre‐electrophoresis prior to separation of proteins, while multiple other (i.e., companion) slab gels were used but without applying the pre‐electrophoresis technique. Separation results were investigated, showing that the application of the pre‐electrophoresis technique had a significant effect on separations particularly for 6% and 9% gels.

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Section: Introductionmentioning

confidence: 99%

Influence of pre‐electrophoresis on protein separations in polyacrylamide gels

2020

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“…The protocol presented here describes the RNase assay. Although this protocol presumes glycerol gradient purified EXOSC10-3xFLAG-containing exosomes as the point of entry into the assay (Domanski and LaCava, 2017), the method should be applicable to any sufficiently pure and concentrated samples.…”

Section: Introductionmentioning

confidence: 99%

“…Anti-FLAG M2 (Sigma-Aldrich, catalog number: F3165 ) antibody conjugated Dynabeads M-270 Epoxy (Thermo Fisher Scientific, Invitrogen™, catalog number: 14302D) (Domanski and LaCava, 2017)…”

Section: Introductionmentioning

confidence: 99%

“…Note: Purify EXOSC10-containing RNA exosomes as described in the complementary protocol, Affinity purification of RNA exosomes from HEK-293 cells (Domanski and LaCava, 2017), or by other means. This assay may also be applied effectively to affinity purified DIS3-3xFLAG as previously described (Domanski et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

“…The RNA exosome-associated EXOSC10 protein is a distributive, 3’–5’ exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al ., 2016) and further detailed in the companion bio-protocol to this one (Domanski and LaCava, 2017). …”

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confidence: 99%

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RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells

Domanski

LaCava

2017

BIO-PROTOCOL

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The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3’–5’ exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al., 2016) and further detailed in the companion bio-protocol to this one (Domanski and LaCava, 2017).

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Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells

Cited by 5 publications

References 16 publications

Influence of pre‐electrophoresis on protein separations in polyacrylamide gels

Influence of pre‐electrophoresis on protein separations in polyacrylamide gels

RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells

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