GFP is remarkably amenable to protein tagging, which is one of the biggest reasons for its unparalleled success. Though it is very well suited for live imaging, for example, as a reporter for TP53 MITE-seq screen [3], other applications result in quenching of GFP fluorescence, which makes it necessary to employ anti-GFP antibodies. Cryopreservation of tissue samples, histological preparations and use of fixatives usually lead to either complete or partial loss of GFP fluorescence making the use anti-GFP antibodies necessary for obtaining reliable results. The detection of GFP-tagged proteins by Western Blotting and investigation of protein-protein interactions via immunoprecipitation also necessitates the use of GFP antibodies. Anti-GFP antibodies are available as polyclonal or monoclonal antibodies and suited for detection of GFP, GFP variants and GFP fusion proteins on western blots, immunoprecipitation, immunohistochemistry, immunocytochemical localization and immunosorbent assays (ELISA).

As the number of applications for anti-GFP antibodies has grown, so has the number of suppliers that provide this reagent. Labome has surveyed formal publications to compile a list of anti-GFP antibodies that have been successfully used in formal articles (Table 1). Based on this survey, a list of major suppliers and a short review of these antibodies are provided in the following section. Table 2 lists the antibodies commonly used for different methods.

Some suppliers also provide anti-GFP single domain antibodies or nanobodies. These antibodies consist of a single variable antibody domain, and are based on the single domain antibodies derived from Camelids. These antibodies are much smaller than regular antibodies, and are finding important uses. Caussinus E et al have pioneered deGradFP (Figure 2), a genetically encoded method for fast depletion of target GFP tagged proteins in any eukaryotic system [231]. This system relies on a single domain antibody fragment against GFP (vhhGFP4) fused to the F-box domain. This fusion protein binds GFP and directs it to the proteasome for degradation. When GFP tagged protein is the only source of a functional protein, gene function can be analyzed without any concerns for interference from any residual protein. Ries at al achieved nanometer spatial resolution for analysis of microtubules, neurons and yeast cells using GFP tagged proteins and small high affinity single domain anti-GFP antibodies [232]. Bhogaraju S et al immunopurified eGFP-tagged SdeA with anti-GFP agarose beads (a GFP nanobody / VHH) from Chromotek for liquid chromatography-mass spectrometric analysis [233]. Lundby A et al used similar GFP-Trap beads from ChromoTek to immunoprecipitate GFP-conjugated proteins from A549 cell lysates [186].

Several Abcam GFP antibodies are commonly cited: ab290 was used in immunohistochemistry [240], western blot [241] and ChIP [242] ; ab13970 in immunocytochemistry [243, 244], immunohistochemistry [245, 246], and immunohistochemistry for scanning electron microscopy [240] ; ab1218 in immunohistochemistry [247] and western blot [241] ; and ab6556 rabbit anti-GFP antibody was used at 1/1000 to perform immunohistochemistry [248]. Ebright RY et al detected the expression of GFP-tagged proteins in mouse paraffin sections with ab183734 [249].

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Figure 1. Left panel: (a-c) Co-injection of DNb-catenin-gfp RNA and MO1-ccr7 or ccr7 RNA showed that Ccr7 can downregulate b-catenin, shown at higher magnification (d-f). Reproduced from Figure 3D of [1]. Right panel: Western blot analysis of co-injection of DNb-catenin-gfp RNA and ccr7 RNA or MO1-ccr7. Reproduced from Fig 3E of [1].

Santa Cruz anti-GFP antibody sc-8334 was used to perform coimmunoprecipitation to study the functions of CCA1 and LHY in regulating the circadian rhythms of Arabidopsis [250] and was used to carry out western blot analysis in order to investigate the role for CHBP protein from Burkholderia pseudomallei as a potent inhibitor of the eukaryotic ubiquitination pathway [251]. And Santa Cruz anti-GFP sc-9996 was used to perform western blot to show that aneuploidy in tumor cells is associated with STAG2 inactivation [252].

Roche mouse monoclonal anti-GFP antibody (clone 7.1/13.1, and also catalog number 11814460001) was used to carry out immunoprecipitation from HEK293T for mass spectrometry analysis to identify nucleolus as a phase-separated protein quality control entity [253]. Mouse monoclonal anti-GFP MAB3580 antibody is a popular choice [95, 254].

H Yu et al detected clover-labeled TDP-43 in immuno electron microscopy with the mouse anti-GFP antibody from Clontech (JL-8) [255]. A Badimon et al used mouse monoclonal anti-GFP antibodies (clones 19C8 and 19F7) from Antibody and Bioresource Core Facility, Memorial Sloan Kettering for immunoprecipitation [256]. A Constantinescu-Bercu et al detected the expression of SLC44A2 tagged with tGFP in HEK293T cells with OriGene clone 2H8 mouse anti-GFP antibody (TA150041) [257]. Nordmann et al detected GFP transfected into pigeon embryonic fibroblasts with 1:500 clone 2B6 antibody from Monoclonal Antibody Facility, Max Perutz Laboratories [258]. Li Z et al detected GFP–LC3B and free GFP (from lysosomal cleavage) in the lysates of HeLa cells stably expressing GFP–LC3B with the Cell Signaling Technologies anti-GFP rabbit monoclonal antibody (a pd:1465972>2956) [259]. Lundby A et al evaluated the GFP expression in A549 cells with the same anti-GFP antibody in Western blots [186]. Chicken antiGFP (1:3000) from Aves Labs (GFP-1020) appear to be a popular choice for immunohistochemical staining [260, 261]. Frottin F et al incubated HEK293T cells with GFP-SOMAmer from SomaLogic to enable DNA-PAINT under super-resolution microscopy to establish nucleolus as a phase-separated protein quality control entity [253].

The application of the anti-GFP antibody is an important factor in deciding the supplier. Suppliers list specific applications for which their antibody is most suitable, and often suggest at what dilutions it may be used. However, performance may vary depending on the specific system and the experimental conditions under which the antibody is used. Many suppliers offer customer support services that may be able to advise on the suitability of their antibody for your applications.

Anti-GFP antibodies recognize other variants of Aequorea victoria GFP.

A lack of signal following western blotting may indicate a few different problems. No or very poor transfer means no signal. The lack of good transfer to the membrane may be checked by Ponceau-S staining. The presence of air bubbles will also result in a poor transfer. The expression level of the GFP-tagged protein may be too low. Load a larger volume of the sample and include a positive control. A very diluted antibody may be the problem, try a few different concentrations of the antibody to probe the western blot. Also, a remote possibility is that the GFP tag is out of frame and is not expressed resulting in a lack of any signal.

GFP fluorescence is either partially or completely lost under certain experimental conditions. Therefore, some cellular structures expressing GFP-tagged protein may be difficult to visualize or image. GFP-Booster is a VHH domain binding protein derived from Camelid that is coupled to a strong fluorescent dye. This stabilizes and enhances the fluorescence signal.

Antibody cross-reactivity is a common problem that usually results in more bands than expected. More stringent experimental conditions may resolve this problem. The GFP-Trap may be used for immunoprecipitation experiments. The GFP-Trap is a single domain antibody (derived from Camelids) with higher specificity, stability and affinity. However, one must consider that if truncated versions of the fusion protein are generated, these will also be detected by the antibody if the tag is present, and result in multiple bands.

Green Fluorescent Protein (GFP) is a small protein from jellyfish, Aequorea victoria. It was discovered by Osamu Shimomura, and was shown to fluoresce green when excited by light in the blue to ultraviolet range. Related fluorescent proteins are found in many other marine organisms, such as corals, anemones and sea pansies. GFP became a very popular tool when it was demonstrated by Martin Chalfie that GFP gene could be cloned and expressed as a luminous tag. Since then, it has been expressed in bacteria, yeast, slime molds, worms, fruit flies, zebrafish, mammalian cell lines and plants. GFP tolerates a wide variety of protein fusions at its N- and C-terminals and does not need any co-factors to fluoresce, which makes it one of the most widely used protein tags. This seminal work opened new avenues for investigating important questions in cell and development biology. The color palette for GFP has been extended beyond green through the efforts of many scientists, in particular that of Roger Tsien’s group. This allows the use of multiple variants to track many biological processes simultaneously. In 2008, Osamu Shimomura, Martin Chalfie and Roger Tsien shared the Nobel Prize in Chemistry for their contributions to the glowing world of GFP.

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Figure 2. deGradFP system (Adapted from ‘Using mutants, knockouts, and trangenesis to investigate gene function in Drosophila. WIRES Developmental biology, 2012, dx.doi.org/10.1002/wdev.101)

GFP forms a very stable barrel-shaped structure that consists of 11 beta sheets surrounding a central alpha helix. The beta sheets are linked through proline-rich loops. The amino acid side chains in each sheet alternately project into the interior of the protein or outward from the surface. The GFP chromophore is formed by the intramolecular cyclization of Ser65, Tyr66 and Gly67, and it sits almost in the center of the barrel [262]. Although this three amino acid motif is common in nature, it does not produce fluorescence. It is the unique internal environment of the GFP beta-barrel that creates the chromophore and also protects it from quenching [263]. The formation of the chromophore is autocatalytic [263]. GFP exists in a predominantly protonated state with an excitation maximum of 395 nm and a less prevalent, non-protonated state that has an excitation peak of 475 nm [264]. The fluorescence emission for both forms peaks at 510 nm.

Since GFP became popular in biosciences, much effort has been devoted to fine-tuning its fluorescence. The jellyfish GFP is quite bright and photostable but its excitation maximum (395 nm) lies in the border of the ultraviolet range. The ultraviolet light damages cellular structures while imaging. A mutagenesis screen uncovered a point mutant, S65T whose excitation maximum is shifted to 488 nm [265]. This variant called ‘EGFP’ circumvents the problem of using ultraviolet light for excitation, and can be easily imaged using the commonly available fluorescein isothiocyanate (FITC) filters. It is also the brightest and the most photostable GFP. However, an important drawback of using GFP is that its spectral behavior is affected by pH. GFP fluorescence gets quenched when it is directed to acidic compartments, such as the endosomes or lysosomes. A photoactivatable variant (PA-GFP) increases fluorescence 100 times when excited by 488-nanometer light after intense irradiation with 413-nanometer light (or by two-photon excitation) and the fluorescence remains stable for days under aerobic conditions [266].

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Figure 3. Regions from DNA-stained squash preparations of Drosophila testes expressing either only Cid-EGFP or only Cenp-C-EGFP. Reproduced from Figure 1 of [2].

Using mutagenesis strategies, the color palette of GFP has been extended beyond green. Blue, cyan and yellow fluorescent proteins (BFP, CFP and YFP respectively), all derivatives of GFP, have proven to be very valuable research tools. As red-shifting the fluorescent properties of GFP beyond yellow has not been successful, researchers explored corals and sea anemones for naturally occurring fluorescent proteins in other colors [267, 268]. Continued efforts have led to other fluorescent proteins with desirable spectral properties that can be used in multicolor labeling, for example, the identification of acid-tolerant GFP protein from Olindias formosa, which can be used in lysosomes [269]. Yang J et al employed a halide-sensitive YFP quenching assay to identify PAC/TMEM206 as a proton-activated chloride channel [270]. pHluorin, a conjugate of a pH-sensitive form of GFP and the VAMP luminal fragment, is a commonly used ratiometric pH sensor [271, 272].

The availability of GFP variants as well as fluorescent proteins from other sources allows researchers to track various biological phenomena simultaneously, perform fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) experiments. One of the most interesting and impressive applications of fluorescent proteins has been the generation of brainbow to study how neuron circuits are organized [273]. Through the random expression of different ratios of red, green and blue variants, the neurons in the brain light up in a variety of colors. In contrast to multi-labeling techniques that limit the use of a few variants, the brainbow is extremely flexible, and can generate up to 100 different hues in individual neurons.