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Figure 2 | BMC Biotechnology

Figure 2

From: A miniaturized sandwich immunoassay platform for the detection of protein-protein interactions

Figure 2

Evaluation of the capability and reproducibility of MSIP. HEK293 cells were cotransfected with FLAG-bait and myc-prey constructs. For negative controls, each myc-prey was cotransfected with pflag-CMV-2. Cell lysates were incubated in the frame of the anti-FLAG spotted slides at room temperature for 1 h. FLAG-bait and any interacting proteins were captured on the surface of the slides. Myc-prey was detected by using monoclonal anti-myc-cy3 (1:200). The image was scanned by a fluorescence scanner, and the NFI of each PPI was calculated. A) Six pairs of well-characterized interacting proteins and 4 pairs of known non-interacting proteins were analyzed by MSIP. The analysis was performed on each of three slides assayed on different days with equal amounts of cell lysates derived from independent transfections. B) eight pairs of protein partners using the same bait were analyzed by MSIP with a slide. Equal amount of cell lysates were incubated on the anti-flag spotted slides. C) Different quantities of the same cell lysates were used to analyze the NFI of two pairs of previously identified positive PPIs by MSIP with two slides.

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