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Epitope-Tagged Protein Immunoprecipitation

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Immunoprecipitation (IP) assays were carried out on plasmid transfected or recombinant Ad transduced fibroblast cells using commercial GFP-trap reagent (ChromoTek) or RFP-trap (ChromoTek) following manufacturer’s protocols and inclusion of protease inhibitor cocktail (Pierce) in cell lysates. Samples were subsequently analyzed by SDS-PAGE (4–20% gradient gel) and western blot using specific anti-epitope tag antibodies: HA (Novus Biologicals); FLAG (Novus Biological); GFP (Santa Cruz Biotechnology); Myc-c (Novus Biologicals); and mCherry (Clontech Laboratories). Appropriate secondary anti-mouse or anti-rabbit HRP conjugate (Cell Signaling Technology) were also used following standard western blot protocol as previously described [36 (link)].

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Coleman S., Choi K.Y., Root M, & McGregor A. (2016). A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus. PLoS Pathogens, 12(7), e1005755.

Publication 2016

GFP-trap reagent RFP-trap FLAG Myc-c mCherry

Anti antibodies Assays Biological Biologicals Cell Epitope Fibroblast cells Immunoprecipitation Mouse Novus Plasmid Protease inhibitor Rabbit Sds page Western blot

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Variable analysis

independent variables

Plasmid transfection
Recombinant Ad transduction

dependent variables

Protein-protein interactions as measured by immunoprecipitation (IP) assays
Protein expression as measured by Western blot

control variables

Use of commercial GFP-trap and RFP-trap reagents (ChromoTek) for immunoprecipitation
Inclusion of protease inhibitor cocktail (Pierce) in cell lysates
Use of specific anti-epitope tag antibodies: HA (Novus Biologicals), FLAG (Novus Biological), GFP (Santa Cruz Biotechnology), Myc-c (Novus Biologicals), and mCherry (Clontech Laboratories)
Use of appropriate secondary anti-mouse or anti-rabbit HRP conjugate (Cell Signaling Technology)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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