High-affinity labeling and tracking of individual histidine-tagged proteins in live cells using Ni2+ tris-nitrilotriacetic acid quantum dot conjugates.

نویسندگان

  • Victor Roullier
  • Samuel Clarke
  • Changjiang You
  • Fabien Pinaud
  • G Géraldine Gouzer
  • Dirk Schaible
  • Valérie Marchi-Artzner
  • Jacob Piehler
  • Maxime Dahan
چکیده

Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation.

Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Di...

متن کامل

Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step.

The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11. This construct was used to transform cells from both Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch. pombe cel...

متن کامل

Probing protein conformations by in situ non-covalent fluorescence labeling.

The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins...

متن کامل

NTA.Ni2+-Functionalized Quantum Dots for VAMP2 Labeling in Live Cells

An efficient method for labeling individual proteins in live cells is required for investigations into biological mechanisms and cellular processes. Here we describe the preparation of small quantum dots (QDs) that target membrane surface proteins bearing a hexahistidine-tag (His6-tag) via specific binding to an nitrilotriacetic acid complex of nickel(II) (NTA·Ni) on the QD surfaces. We showed ...

متن کامل

Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve sin...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Nano letters

دوره 9 3  شماره 

صفحات  -

تاریخ انتشار 2009