Schulte R et al. (2008), Single bead affinity detection (SINBAD) for the...

XB-ART-37821

PLoS One 2008 Apr 09;34:e2061. doi: 10.1371/journal.pone.0002061.

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Single bead affinity detection (SINBAD) for the analysis of protein-protein interactions.

Schulte R , Talamas J , Doucet C , Hetzer MW .

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We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment.

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Species referenced: Xenopus
Genes referenced: ahctf1 nup107 nup133 nup160 nup37 nup43 nup85 nup98 sec13 sln terf2

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	Figure 1. Monitoring protein interactions on a single affinity chromatography bead.(A) 1 µM recombinant his-TAP-tagged Importin β(wt) was immobilized on 5 ul magnetic Ni-NTA beads for 20 min. After the addition of 5 nM CdSe-ZnS core shell QD655 beads were washed and isolated with a magnet. Beads were recovered in 2 ul PBS, mounted on a glass slide and the bead surface was imaged by confocal microscopy. (B) Schematic illustration of the RanGTP (gray) dissociation of Importin β (red) from Importin α (light blue). (c) QD585 cross-linked to NLS peptide (blue), QD705 cross-linked to reverse NLS (SLN) (magenta), streptavidin-coated QD655-TAP-Importin β(wt) (red) and streptavidin-coated QD605-TAP-Importin β(ΔN44) (green) were incubated with his-Importin α coated Ni-NTA beads in the presence of buffer (control), 20 mM NLS or SLN peptides or 10 mM RanQ69L.
	Figure 2. Using SINBAD as pull-down method with increased sensitivity.(A) Approx. 10 Ni-NTA beads were incubated with reticulocyte lysates containing his-Importin α or his- Importin α together with in vitro translated TAP-Importin β(wt) in the presence or absence of excess Importin α. QD655 were added, beads were isolated and imaged. (B) his-Nup160 was translated in reticulocyte lysates and incubated with equal volume (2 ul) of in vitro translated Nup133-TAP, Nup107-TAP, Nup96-TAP, Nup85-TAP, Nup43-TAP, Nup37-TAP, Seh1-TAP, Sec13-TAP and ELYS-TAP for 30 min. Approx. 5 Ni-NTA beads were added together with streptavidin-coated QDs655 and imaged by confocal microscopy on the bead surface. (C) Schematic illustration of SINBAD for the pull-down of endogenous proteins. (D) Hypotonic cell lysates corresponding to 500 or 50 cells were incubated with Ni-NTA beads coated with his-RanQ69L. Bound endogenous Importin β was visualized as described in (C). (E) Random 10 µm2 areas were imaged and fluorescence intensity at 655 nm was plotted. n = 20.
	Figure 3. SINBAD for the detection of protein-DNA interactions.(A) his-TRF2 was immobilized on Ni-NTA beads and incubated with hybridized 5′-biotinylated DNA oligos containing three repeats of TTAGGG (red), a mutant TTACGG or ssTTAGG as indicated. Bound DNA was visualized with streptavidin-coated QDs655. (B) <10 his-TRF2 Ni-NTA beads bound to wild-type telomere sequence were incubated with increasing concentrations of competitor DNA oligos. Fluorescence intensity of QD655 was determined from confocal images and plotted.

References [+] :

Anderson, Nuclear envelope formation by chromatin-mediated reorganization of the endoplasmic reticulum. 2007, Pubmed, Xenbase