GFP circularization assays were performed in a manner similar to that previously described (Strijbis et al. 2012 (link)). In short, HEK293T cells were transfected with pcDNA3.1-Gly-GFP-LPETG-Myc along with empty pUC19 (Control), pcDNA3.1-HA-S. pyogenese SrtA, or pcDNA3.1-HA-R15-78 using Lipofectamine2000 according to manufacturer instructions. 24 hrs later the cells were lysed in 1X SDS-PAGE loading dye, boiled for 10 min, and run on a 12% polyacrylamide gel. The gel was then transferred onto nitrocellulose paper and probed with anti-GFP-HRP (Stefanovic and Hegde 2007 (link)) or an anti-HA (Abcam 18181) followed by anti-mouse or anti-rabbit horseradish peroxidase secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). The blots were then incubated with SuperSignal West Pico (Pierce, Rockford Il) chemiluminescent substrate according to manufacturer instructions and exposed on Crystalgen blue sensitive film.
Protocol detail
GFP Circularization Assay in HEK293T Cells
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Anti antibodies
Assays
Cells
Horseradish peroxidase
Mouse
Nitrocellulose
Polyacrylamide gel
Rabbit
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Corresponding Organization : Albert Einstein College of Medicine
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Variable analysis
independent variables
- Transfection with pcDNA3.1-Gly-GFP-LPETG-Myc along with empty pUC19 (Control)
- Transfection with pcDNA3.1-HA-S. pyogenese SrtA
- Transfection with pcDNA3.1-HA-R15-78
- GFP circularization
- Expression of HA-tagged proteins (S. pyogenese SrtA and R15-78)
- Cell type (HEK293T cells)
- Transfection method (Lipofectamine2000)
- Lysis and gel electrophoresis conditions
- Antibodies used for detection (anti-GFP-HRP, anti-HA)
- Positive control: Transfection with pcDNA3.1-Gly-GFP-LPETG-Myc along with empty pUC19
- Negative control: Not explicitly mentioned
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