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 More genes are active in high-performance maize
19.01.2018 | Rheinische Friedrich-Wilhelms-Universität Bonn

nachricht How plants see light
19.01.2018 | Albert-Ludwigs-Universität Freiburg im Breisgau

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: Artificial agent designs quantum experiments

On the way to an intelligent laboratory, physicists from Innsbruck and Vienna present an artificial agent that autonomously designs quantum experiments. In initial experiments, the system has independently (re)discovered experimental techniques that are nowadays standard in modern quantum optical laboratories. This shows how machines could play a more creative role in research in the future.

We carry smartphones in our pockets, the streets are dotted with semi-autonomous cars, but in the research laboratory experiments are still being designed by...

Im Focus: Scientists decipher key principle behind reaction of metalloenzymes

So-called pre-distorted states accelerate photochemical reactions too

What enables electrons to be transferred swiftly, for example during photosynthesis? An interdisciplinary team of researchers has worked out the details of how...

Im Focus: The first precise measurement of a single molecule's effective charge

For the first time, scientists have precisely measured the effective electrical charge of a single molecule in solution. This fundamental insight of an SNSF Professor could also pave the way for future medical diagnostics.

Electrical charge is one of the key properties that allows molecules to interact. Life itself depends on this phenomenon: many biological processes involve...

Im Focus: Paradigm shift in Paris: Encouraging an holistic view of laser machining

At the JEC World Composite Show in Paris in March 2018, the Fraunhofer Institute for Laser Technology ILT will be focusing on the latest trends and innovations in laser machining of composites. Among other things, researchers at the booth shared with the Aachen Center for Integrative Lightweight Production (AZL) will demonstrate how lasers can be used for joining, structuring, cutting and drilling composite materials.

No other industry has attracted as much public attention to composite materials as the automotive industry, which along with the aerospace industry is a driver...

Im Focus: Room-temperature multiferroic thin films and their properties

Scientists at Tokyo Institute of Technology (Tokyo Tech) and Tohoku University have developed high-quality GFO epitaxial films and systematically investigated their ferroelectric and ferromagnetic properties. They also demonstrated the room-temperature magnetocapacitance effects of these GFO thin films.

Multiferroic materials show magnetically driven ferroelectricity. They are attracting increasing attention because of their fascinating properties such as...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

10th International Symposium: “Advanced Battery Power – Kraftwerk Batterie” Münster, 10-11 April 2018

08.01.2018 | Event News

See, understand and experience the work of the future

11.12.2017 | Event News

Innovative strategies to tackle parasitic worms

08.12.2017 | Event News

 
Latest News

More genes are active in high-performance maize

19.01.2018 | Life Sciences

How plants see light

19.01.2018 | Life Sciences

Artificial agent designs quantum experiments

19.01.2018 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>