Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Suitable Protein Tags for Nanoscopy

29.01.2016

Scientists of the Göttingen Cluster of Excellence CNMPB and the EMBL describe efficient fluorescence tag for super-resolution microscopy. Published in ACS Nano.

Modern optical technologies such as super-resolution nanoscopy enable to exactly image small structures and molecular processes, therefore, providing a fascinating view into living cells. To visualize such processes in the nanometer range, cellular structures of interest have to be efficiently labeled.


Protein labeling with aptamers (left) better displays the shape of endosomes than labeling with larger antibody probes (right).

Felipe Opazo / CNMPB

Fluorescent proteins are routinely used as convenient tags in conventional microscopy but their use for nanoscopy has been questioned. Scientists of the Göttingen Cluster of Excellence and DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) and of the EMBL in Heidelberg now investigated the application of fluorescent protein tags on the organization of proteins.

The findings of the team around Nobel Prize awardee Stefan W. Hell (Max Planck Institute for Biophysical Chemistry in Göttingen), Edward Lemke (EMBL, Heidelberg) and Silvio O. Rizzoli (University Medical Center Göttingen) proved the general use of fluorescent protein tags to be reliable tools in nanoscopy and for biomedical research.

The results, recently published in ACS Nano, specially indicate fluorescence labeling based on unnatural amino acids to be a reliable alternative for labeling.

Original publication: Vreja IC, Nikić I, Göttfert F, Bates M, Kröhnert K, Outeiro TF, Hell SW, Lemke EA, Rizzoli SO (2015) Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein tagging on Protein Assemblies. ACS NANO, 9(11): 11034-41.

Nanoscopy, or lens-based super-resolution microscopy, is a fairly new technique that enables to observe molecules and biomolecules with a resolution of 10-30 nm and to moreover analyze whole protein complexes. For their development and the implementation of super-resolution nanoscopy coauthor Stefan W. Hell, has been awarded with the Nobel Prize in Chemistry 2014 together with Betzig and William E. Moerner. For labeling and visualization procedures fluorescent protein tags are used.

Due to their relatively large size these labels often tend to form clusters, thus, producing artifacts in nanoscopic recordings. „Regarding the ease of use and their compatibility, we are specially interested in genetically encoded fluorescent protein tags, which can be introduced into living cells by gene manipulation. Their use in nanoscopy should therefore be carefully investigated“, senior author Silvio O. Rizzoli explains.

The scientists focused on 26 proteins, which are known to form various types of multi molecular arrangements and compared their nanoscale organization with or without fluorescent tag. The proteins were labeled with the smallest tag that is currently available, namely the unnatural amino acid propargyl-L-lysine (PRK) by insertion into the coding sequence of a target protein.

For visualization with a super-resolution microscope, synthetic fluorophores were coupled to the PRK via “click chemistry”. This fast and selective organic reaction is an established a standard tool for labeling and modification of biomolecules.

The researchers subsequently compared the nanoscopic organization of labeled and non-labeled proteins using ground-state depletion followed by individual molecule return microscopy (GSDIM) and stimulated emission depletion (STED) microscopy. Apart from six proteins that turned out to be slightly adversely affected, the FP-tagged and non-FP-tagged proteins formed similar molecular arrangements. In total, the scientists assessed the use of unnatural amino acids as suitable markers for nanoscopy.

“Other research groups should now also test this labeling technique to see whether their target proteins are affected by the fluorescent protein tags or not,” first author Ingrid Vreja recommends. Together with their findings, the authors suggest an easily implementable detailed protocol that other researchers may use for testing and/or labeling their own proteins of interest.

Weitere Informationen:

http://www.rizzoli-lab.de Homepage of the Department of Prof. Dr. Silvio O. Rizzoli
http://www.cnmpb.de CNMPB - Cluster of Excellence and DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain

Dr. Heike Conrad | idw - Informationsdienst Wissenschaft

More articles from Life Sciences:

nachricht Could this protein protect people against coronary artery disease?
17.11.2017 | University of North Carolina Health Care

nachricht Microbial resident enables beetles to feed on a leafy diet
17.11.2017 | Max-Planck-Institut für chemische Ökologie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: A “cosmic snake” reveals the structure of remote galaxies

The formation of stars in distant galaxies is still largely unexplored. For the first time, astron-omers at the University of Geneva have now been able to closely observe a star system six billion light-years away. In doing so, they are confirming earlier simulations made by the University of Zurich. One special effect is made possible by the multiple reflections of images that run through the cosmos like a snake.

Today, astronomers have a pretty accurate idea of how stars were formed in the recent cosmic past. But do these laws also apply to older galaxies? For around a...

Im Focus: Visual intelligence is not the same as IQ

Just because someone is smart and well-motivated doesn't mean he or she can learn the visual skills needed to excel at tasks like matching fingerprints, interpreting medical X-rays, keeping track of aircraft on radar displays or forensic face matching.

That is the implication of a new study which shows for the first time that there is a broad range of differences in people's visual ability and that these...

Im Focus: Novel Nano-CT device creates high-resolution 3D-X-rays of tiny velvet worm legs

Computer Tomography (CT) is a standard procedure in hospitals, but so far, the technology has not been suitable for imaging extremely small objects. In PNAS, a team from the Technical University of Munich (TUM) describes a Nano-CT device that creates three-dimensional x-ray images at resolutions up to 100 nanometers. The first test application: Together with colleagues from the University of Kassel and Helmholtz-Zentrum Geesthacht the researchers analyzed the locomotory system of a velvet worm.

During a CT analysis, the object under investigation is x-rayed and a detector measures the respective amount of radiation absorbed from various angles....

Im Focus: Researchers Develop Data Bus for Quantum Computer

The quantum world is fragile; error correction codes are needed to protect the information stored in a quantum object from the deteriorating effects of noise. Quantum physicists in Innsbruck have developed a protocol to pass quantum information between differently encoded building blocks of a future quantum computer, such as processors and memories. Scientists may use this protocol in the future to build a data bus for quantum computers. The researchers have published their work in the journal Nature Communications.

Future quantum computers will be able to solve problems where conventional computers fail today. We are still far away from any large-scale implementation,...

Im Focus: Wrinkles give heat a jolt in pillared graphene

Rice University researchers test 3-D carbon nanostructures' thermal transport abilities

Pillared graphene would transfer heat better if the theoretical material had a few asymmetric junctions that caused wrinkles, according to Rice University...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Ecology Across Borders: International conference brings together 1,500 ecologists

15.11.2017 | Event News

Road into laboratory: Users discuss biaxial fatigue-testing for car and truck wheel

15.11.2017 | Event News

#Berlin5GWeek: The right network for Industry 4.0

30.10.2017 | Event News

 
Latest News

NASA detects solar flare pulses at Sun and Earth

17.11.2017 | Physics and Astronomy

NIST scientists discover how to switch liver cancer cell growth from 2-D to 3-D structures

17.11.2017 | Health and Medicine

The importance of biodiversity in forests could increase due to climate change

17.11.2017 | Studies and Analyses

VideoLinks
B2B-VideoLinks
More VideoLinks >>>