Antibodies Against Fluorescent Proteins

Fluorescent proteins such as GFP, RFP, and their derivatives are indispensable tools for visualizing and tracking biological processes across diverse experimental systems. To fully exploit their potential, reliable and application-validated detection and capture reagents are essential. Our portfolio of antibodies against fluorescent proteins is designed to support both analytical and preparative workflows, ranging from flow cytometry (FACS), flow cytometry–based sorting, and immunohistochemistry (IHC), to robust affinity-based capture strategies.

In addition to conventional detection antibodies, we offer a dedicated catcher product line: high-affinity antibodies optimized for pull-down applications, including immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP). Our catcher product line is based on recombinant single-domain antibodies (sdAbs) derived from llamas or alpacas. These reagents enable efficient and specific isolation of fluorescent protein–tagged targets, facilitating downstream biochemical and molecular analyses with minimal background.

All products are developed with a strong emphasis on specificity, reproducibility, and batch-to-batch consistency, supported by rigorous quality control and application-relevant validation. Learn more about the history of fluorescent proteins down below, how different variants emerged and about their characteristics; advantages and disadvantages for specific usecases.

History of Green Fluorescent Protein (GFP)

The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria which fluoresces green upon excitation with blue or ultraviolet light. The discovery of green fluorescent protein in the early 1960s by Shimomura has laid the foundation for modern live cell imaging. GFP can be fused to other proteins of interest in a host cell as a noninvasive fluorescent marker in living cells and organisms. Since then, GFP has been engineered and further developed to produce a plethora of mutants coverving a wide range of colors, including enhanced green, cyan and yellow fluorescent proteins, which exhibit peak emission wavelengths ranging from 425 to 525 nm.

Comparison of GFP Variants

Because the construction of red-shifted mutants from the Aequorea victoria jellyfish GFP beyond the yellow spectrum proved largely unsuccessful, researchers have searched for additional options. In 1999 the red fluorescent protein DsRed, derived from Discosoma sea anemones was mentioned first in a publication by Matz et al. Once fully matured, the fluorescence emission spectrum of DsRed features a peak at 583 nm whereas the excitation spectrum has a major peak at 558 nm and a minor peak around 500 nm.

Fluorescent Protein: GFP vs RFP

In comparison to GFP, wild-type DsRed shows certain downfalls but also some unique benefits. Unlike GFP, the fluorescence of DsRed remains stable in acidic pH ranges. Furthermore, DsRed is particulary well suited for fluorescence microscopy, due to its good contrast. Additionally, it has a high quantum yield and is photo stable. On the other hand, maturation of DsRed fluorescence occurs slowly over a time frame of 24 hours. Thus, it cannot be used to monitor proteins with a short halflife. DsRed is an obligate tetramer and can form large protein aggregates in living cells which pose a higher risk for toxicity. Overall, GFP can successfully conjugate with a broader variety of proteins.

Catcher - High-Affinity Single-Domain Antibodies

antibodies-online offers catchers, the ideal tool for effective pulldown of GFP, RFP or BFP fusion proteins. Our catcher product lines are based on recombinant single-domain antibodies (sdAbs) derived from llamas or alpacas. This type of affinity molecules provide significant advantages over conventional IgG molecules. All of our products are produced in-house. They are highly specific with affinities tailored for a wide range of applications.

RFP Variants

Similar to GFP several RFP variants were engineered to improve its characteristics in general or optimize for specific experiments. DsRed2 contains several mutations at the N-terminus that prevent formation of protein aggregates and reduce toxicity. Red fluorescence emission from DsRed-Express can be observed within an hour after expression, 11 times faster than DsRed. DsRed-Express is a precursor RFP of more recent derivatives including tdTomato and the monomeric mCherry, mOrange, and mStrawberry.

Comparison of RFP Variants

mCherry is together with mOrange and mStrawberry a member of the mFruits family of monomeric red fluorescent proteins. In comparison to the progenitor DsRed, mOrange, mStrawberry and mCherry produces strong blue- and red-shifted variants. They have a lower molecular weight and will fold faster than tetramers do and therefore disturb the target system less. Out of all of the true monomers developed, mCherry has the longest wavelengths, the highest photostability, fastest maturation and excellent pH resistance. mOrange overcomes mCherry in quantum yield however it does exhibit substantial acid sensitvity. A high extinction coefficient and quantum yield makes the large stoke shift variant LSSmOrange (in combination with T-Sapphire) a suitable candidate for FRET applications.

tdTomato is a genetic fusion of two copies of the dTomato gene which was specifically designed for low aggregation. Its tandem dimer structure plays an important role in the exceptional brightness of tdTomato, which is 5 times higher compared to EGFP. tdTomato's emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. Because tdTomato forms an intramolecular dimer, it behaves like a monomer and is as photostable as mCherry.

Phototransformable Fluorescent Proteins

Dendra2 is an improved version of the green-to-red photoswitchable fluorescent protein Dendra, derived from octocoral Dendronephthya species. Dendra2 exhibits faster maturation and brighter fluorescence both before and after photoswitching than that of Dendra. In contrast to all other monomeric photoactivatable FPs, which necessarily require UV-violet (e.g., 405 nm laser) light for activation, Dendra and Dendra2 permit the use of blue (e.g., 488 nm laser) activating light. The phototransformative property has allowed highlighting and tracking of subpopulations of cells, organelles, and proteins in living systems. With Dendra2 newly synthesized proteins that are en route to their final destinations can be visualized.

Further Reading

• An Introduction to Fluorescence (Part 1)
• An Introduction to Fluorescence (Part 2)
• Excitation / Emission Maxima Poster for Popular Fluorophores

References