Recently a new bright monomeric yellow-green fluorescent protein has been published, which is called mNeonGreen. This protein has already been frequently used for mainly microscopic applications in both wide-field microscopy and super resolution microscopy. What is mNeonGreen all about?
mNeonGreen vs. GFP – What are the differences?
Origin of mNeonGreen
GFP has been originally isolated from jellyfish Aequorea Victoria. Frequently, it has been genetically modified to generate a large number of GFP variants (derivatives) with special spectral or functional properties. The most common variant EGFP has an excitation peak at 488 nm and an emission peak at 509 nm. The filter settings of most microscopes are optimized for those wavelengths.
Evolutionary distance of mNeonGreen to jellyfish fluorescent proteins
mNeonGreen is not a variant of GFP. Instead, mNeonGreen is derived from the lancelet Branchiostoma lanceolatum multimeric yellow fluorescence protein. Hence, mNeonGreen is evolutionarily distant from jellyfish-derived fluorescent proteins. At sequence level, mNeonGreen shares just 20-25% sequence identity with common GFP derivatives:
Comparison of EGFP(1) and mNeonGreen(2) amino acid sequences
The mNeonGreen DNA sequence can be found here: https://www.ncbi.nlm.nih.gov/nuccore/KC295282.1 or https://www.ebi.ac.uk/ena/data/view/KC295282
Due to the low sequence similarity to mNeonGreen, it is expected that in general affinity tools for GFP variants, i.e. antibodies, should not bind to mNeonGreen and vice versa.
Excitation & Emission wavelengths and brightness of mNeonGreen
Furthermore, mNeonGreen has an excitation maximum at 506 nm and an emission maximum at 517 nm (Shaner et al., 2013). mNeonGreen is compatible with most GFP filter sets. Apparently, it is 3 times brighter than GFP when using GFP filters; optimization of filters may increase its brightness further.
In addition, mNeonGreen seems to be more stable and less sensitive to laser induced bleaching than EGFP. Therefore, mNeonGreen is particular suitable for confocal and super resolution microscopy, especially when fusion proteins are investigated, which are expressed at low levels.
Size and Structure of mNeonGreen
mNeonGreen consists of 237 amino acids, which translates into 26.6 kDa molecular weight. This size, but not sequence, is very similar to EGFP that comprises 239 amino acids and is 26.9 kDa. The mNeonGreen crystal structure is to our knowledge neither known nor published yet (at publishing date of blog).
Expression of mNeonGreen in multicellular organisms like C.elegans
Hostettler et al., 2017, analyzed and compared the expression pattern of GFP-fusion proteins with mNeonGreen-fusion proteins in C.elegans. They concluded: “While mNeonGreen might be equivalent to GFP for strong expression levels, it represents a valuable alternative, for example, in a number of non-embryonic tissues examined in the present study”, and “As a whole, our work established mNeonGreen as a brighter alternative to GFP in vivo imaging in a multicellular organism.”
Applications of GFP and mNeonGreen
Since its introduction as a molecular biology tool in the early 1990s, GFP and its variants have been extensively used for a multitude of applications such as
- Immunofluorescence (wide field, confocal & super resolution microscopy)
- Immunoprecipitation (IP)/ Co-IP
- Mass spectrometry (MS)
- Enzyme activity measurements
- RIP/ ChIP analysis
- Reporter assays
In order to deal with this variety of experimental options, dedicated research tools have been invented: these include anti-GFP antibodies, GFP-Traps, GFP-Booster, etc. In addition, applications optimized GFP derivatives have been generated: these comprise modified spectral properties, e.g. large stokes shift, split variants, pH sensitive, fast folding, redox sensitive, photoactivatable, etc. variants. Hence, GFP itself has been cited more than 34.000 times in pubmed.
So far, mNeonGreen is mainly used for expression as fusion protein in cells, tissues, or organs and analysis thereof in wide field, confocal & super resolution microscopy. mNeonGreen seems to be more stable in STED and allows to take more images. Its expression pattern is identical or very similar for strongly expressed proteins (Hostettler et al., 2017).
Following the role model GFP, which started as microscopy tool that moved into additional biochemical applications, ChromoTek is proud to launch novel affinity tools to expand mNeonGreen’s applicability to:
- Immunofluorescence (IF)
Anti-mNeonGreen antibody 32F6 (mouse monoclonal)
- Immunoprecipitation (IP)
mNeonGreen-Trap (single domain antibody (sdAb, nanobody, or VHH) from alpaca coupled to agarose or magnetic agarose)
For biochemical analysis, including mass spectrometry and enzyme activity measurements, mNeonGreen-fusion proteins and their interacting factors can be fast and efficiently isolated by immunoprecipitation using the mNeonGreen-Trap.
- Western blot (WB)
Anti-mNeonGreen antibody 32F6 (mouse monoclonal)
Anti-mNeonGreen antibody 32F6 (mouse monoclonal) and mNeonGreen-Trap (sdAb from alpaca coupled to agarose or magnetic agarose)
So far, mNeonGreen has been cited 12 times in PubMed at the date of writing this blog. In Google scholar there are already about 300 hits, including some that may not be related.
Now, ChromoTek offers the first anti-mNeonGreen antibody for immunofluorescence and Western blot, to our knowledge: This is the mouse monoclonal, subtype IgG2a anti-mNeonGreen antibody 32F6. It is validated for IF, WB and ELISA.
What is the affinity of anti-mNeonGreen antibodies?
ChromoTek has validated the binding of the anti-mNeonGreen antibody 32F6: it has a high affinity with a dissociation constant KD of just 3.5 nM. In addition, the ChromoTek mNeonGreen-Trap is validated and thoroughly characterized for immunoprecipitation of mNeonGreen-fusion proteins and their interacting partners. With a dissociation constant of KD of 2nM, the ChromoTek mNeonGreen-Trap is apparently the highest affinity resin for mNeonGreen pull-down available.
anti-mNeonGreen antibody 32F6 and mNeonGreen-Trap are catalogue products of ChromoTek and can be purchased under our standard terms and conditions of sale.
Request a free test sample and see for yourself!
N.C. Shaner et al., A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods. 2013 May;10(5):407-9. doi: 10.1038/nmeth.2413. Epub 2013 Mar 24.
Hostettler et al., The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo, G3: Genes, Genomes, Genetics February 1, 2017 vol. 7 no. 2 607-615; https://doi.org/10.1534/g3.116.038133