Protein-protein interaction assay

A GST pull-down assay was performed as previously described [57 (link)]. The Pns11 gene of RGDV was cloned in the pGEX-3x vector to construct a plasmid expressing the GST fusion protein as a bait (GST-Pns11). The full-length ORF of the VDAC from R. dorsalis was cloned into the pHM4 vector to construct a plasmid expressing the His fusion protein as a prey (His-VDAC). Recombinant proteins GST-Pns11 and GST were respectively expressed in the Escherichia coli stain BL21. Lysates were then incubated with glutathione-Sepharose beads (Amersham) and subsequently, with the recombinant protein His-VDAC. Finally, eluates were analyzed using GST-tag and His-tag antibodies (Sigma), respectively, in a Western blot assay.

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Chen Q., Zheng L., Mao Q., Liu J., Wang H., Jia D., Chen H., Wu W, & Wei T. (2019). Fibrillar structures induced by a plant reovirus target mitochondria to activate typical apoptotic response and promote viral infection in insect vectors. PLoS Pathogens, 15(1), e1007510.

Publication 2019

glutathione-Sepharose beads GST-tag His-tag antibodies

Antibodies Assay Escherichia coli Gene Glutathione Plasmid Protein a Recombinant protein Sepharose Stain Vector Western blot assay

Corresponding Organization : Hunan Academy of Agricultural Sciences

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Variable analysis

independent variables

Expression of the GST fusion protein (GST-Pns11) as a bait
Expression of the His fusion protein (His-VDAC) as a prey

dependent variables

Binding between the GST-Pns11 bait and the His-VDAC prey, as detected by Western blot

control variables

Expression of the GST protein alone as a control

controls

Positive control: Incubation of GST-Pns11 with His-VDAC
Negative control: Incubation of GST alone with His-VDAC

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