GFP (green fluorescent protein): Properties, origin, specifications, tips

Everything you need to know about GFP: properties and applications

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Summary

• GFP (green fluorescent protein) is a fluorescent protein originally discovered in the jellyfish Aequorea victoria and is widely used as a live-cell reporter and protein tag in research.
• GFP works as a fusion tag — by genetically attaching GFP to a protein of interest, researchers can track its location, movement, and interactions in real time using fluorescence microscopy.
• EGFP (enhanced GFP) is the most commonly used derivative, with an excitation peak at 488 nm and emission at 507 nm, and a molecular weight of 26.9 kDa.
• GFP and its derivatives are widely used across applications including fluorescence microscopy, reporter assays, immunoprecipitation, protein purification, and FRET-based signal transduction studies.
• Anti-GFP Nanobodies like the GFP-Trap offer a highly specific and low-background method for pulling down GFP-tagged proteins in immunoprecipitation experiments.

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What is GFP?

GFP stands for green fluorescent protein. GFP is a fluorescent protein that can be expressed in vivo. If GFP is exposed to light, it emits a green fluorescent signal. This property has had an enormous impact on cell biology by enabling the imaging of almost any protein, in transcription studies by working as a reporter gene, and in biochemical applications.

Origin of GFP

GFP is an endogenous protein from the jellyfish Aequorea Victoria. It was isolated by Osamu Shimomura in 1962. In 1992, the sequence of GFP was cloned (Douglas Prasher) and Martin Chalfie’s lab expressed the sequence in vivo. Roger Tsien’s lab improved GFP and managed to convert it into a commonly used research tool. In 2008, The Nobel Prize in Chemistry was awarded “for the discovery and development of the green fluorescent protein, GFP.” See Roger Tsien’s Nobel Prize lecture here.

How does GFP work?

GFP is a protein and, like other proteins, it can be expressed by living organisms. Once GFP is expressed and properly folded, it shows fluorescent properties. If excited by light in the ultra-violet or blue spectrum, GFP emits green light (for more details please see sections 8 and 9). This property has had an enormous impact on cell biology: GFP and proteins fused to GFP can be detected as GFP works as a fluorescent tag. This has actually made lots of new experiments possible.

What are GFP-tagged proteins and GFP-fusion proteins

Endogenous proteins do not contain protein or peptide tags and therefore are sometimes difficult to detect in an assay. One solution that enables easy detection is to genetically fuse protein and peptide tags to the protein of interest. Tagged proteins can be used for purposes such as immunoprecipitation, microscopy, protein purification, Western blotting, protein arrays, etc. Proteins fused to GFP are called GFP-tagged proteins or GFP-fusion proteins. GFP-tagged proteins are often used for fluorescence microscopy, immunoprecipitation, protein purification, and Western blotting.

GFP emission spectrum and excitation peaks

Excitation max	Emission max
GFP	395 nm and 475 nm	509 nm
EGFP (most common derivative)	488 nm	507 nm

For more details see https://www.fpbase.org/protein/avgfp/ and https://www.fpbase.org/protein/egfp/

Which applications can GFP be used for?

GFP has been used in many different applications such as:

• imaging of proteins (epi-fluorescent microscopy, confocal microscopy, super-resolution microscopy)
• reporter assays (transcription studies)
• signal transduction studies (FRET: fluorescence resonance energy transfer)
• biochemical applications (immunoprecipitation, protein purification)
• a bio-sensor (pH, calcium)

GFP derivatives

Many fluorescent proteins are based on the GFP sequence. These fluorescent proteins are genetically engineered and have different properties such as different excitation/emission spectra, photo or pH-stability, folding properties, half-time etc. Here is a rough overview:

BFP	GFP Envy	Superfolder GFP (sfGFP)
CFP	GFP S65T	yeast EGFP
AcGFP	GFPSpark	Citrine
Clover	GFPuv	Ecitrine
EGFP	mClover (Clover A260K)	EYFP
Emerald	mEGFP	Venus
G3GFP	mGFP (monomeric GFP)	YFP
GFP	Monomeric EGFP A206K	Ypet
GFP (cycle 3)	mPhluorin
GFP5	PA-GFP

See https://www.fpbase.org/protein/avgfp/ for the complete list.

Structure of GFP

GFP and GFP derivate EGFP have a β-barrel structure. In the center of this β-barrel, there are 3 amino acids. These acids and the cyclization and oxidation of their backbone form a two-ring chromophore.

Left: beta-barrel structure and fluorescent core of enhanced GFP (EGFP), a GFP derivate; right: magnification of the fluorescent core, the two-ring chromophore

Why is GFP fluorescent?

The two-ring chromophore of GFP absorbs and emits light, e.g., light photons, in the visible green spectrum. The chromophore of GFP lies in the center of a beta-barrel structure. The two-ring chromophore is formed by oxidation and cyclization of the backbone of 3 amino acids: Threonine 65, Tyrosine 66, and Glycine 67. This process occurs during the folding of the protein and depends on different factors such as pH, temperature, and oxygen concentration.

Properties and Size of EGFP

Size:
Number of amino acids: 239
Molecular weight (MW): 26,9 kDa

Properties:
Extinction coefficient (EC): 55,900 M-1 cm-1
Maturation rate (at 37°C): 25 min

EGFP Sequences

EGFP amino acid sequence:

10	20	30	40	50
MVSKGEELFT	GVVPILVELD	GDVNGHKFSV	SGEGEGDATY	GKLTLKFICT
60	70	80	90	100
TGKLPVPWPT	LVTTLTYGVQ	CFSRYPDHMK	QHDFFKSAMP	EGYVQERTIF
110	120	130	140	150
FKDDGNYKTR	AEVKFEGDTL	VNRIELKGID	FKEDGNILGH	KLEYNYNSHN
160	170	180	190	200
VYIMADKQKN	GIKVNFKIRH	NIEDGSVQLA	DHYQQNTPIG	DGPVLLPDNH
210	220	230
YLSTQSALSK	DPNEKRDHMV	LLEFVTAAGI	TLGMDELYK

Source: https://www.uniprot.org/uniprot/C5MKY7

EGFP DNA sequences:

atggtgagca	agggcgagga	gctgttcacc	ggggtggtgc	ccatcctggt	cgagctggac	60
ggcgacgtaa	acggccacaa	gttcagcgtg	tccggcgagg	gcgagggcga	tgccacctac	120
ggcaagctga	ccctgaagtt	catctgcacc	accggcaagc	tgcccgtgcc	ctggcccacc	180
ctcgtgacca	ccctgaccta	cggcgtgcag	tgcttcagcc	gctaccccga	ccacatgaag	240
cagcacgact	tcttcaagtc	cgccatgccc	gaaggctacg	tccaggagcg	caccatcttc	300
ttcaaggacg	acggcaacta	caagacccgc	gccgaggtga	agttcgaggg	cgacaccctg	360
gtgaaccgca	tcgagctgaa	gggcatcgac	ttcaaggagg	acggcaacat	cctggggcac	420
aagctggagt	acaactacaa	cagccacaac	gtctatatca	tggccgacaa	gcagaagaac	480
ggcatcaagg	tgaacttcaa	gatccgccac	aacatcgagg	acggcagcgt	gcagctcgcc	540
gaccactacc	agcagaacac	ccccatcggc	gacggccccg	tgctgctgcc	cgacaaccac	600
tacctgagca	cccagtccgc	cctgagcaaa	gaccccaacg	agaagcgcga	tcacatggtc	660
ctgctggagt	tcgtgaccgc	cgccgggatc	actctcggca	tggacgagct	gtacaagtaa	720

Source: https://www.ebi.ac.uk/ena/browser/view/ACS32473

Anti-GFP antibodies and Nanobodies

There are different polyclonal and monoclonal antibodies and an anti-GFP Nanobody available commercially:

• Immunoprecipitation: ChromoTek’s GFP Nanobody GFP-Trap
  • Very low background
  • No dissociating heavy and light antibody chains
  • Stringent washing
• Immunofluorescence: GFP-Booster
  • Better tissue penetration
  • Higher resolution
• Western blotting: GFP antibody [3H9], rat monoclonal
• Immunofluorescence, Western blotting: GFP antibody [PABG1], rabbit polyclonal

GFP beads

Chromotek offers GFP Nanobodies conjugated to beads for immunoprecipitation and unconjugated GFP Nanobodies/VHHs:

GFP-Trap Agarose: anti-GFP Nanobody conjugated to agarose beads
GFP-Trap Magnetic Agarose: anti-GFP Nanobody conjugated to magnetic agarose beads
GFP-Trap Magnetic Particles M-270: anti-GFP Nanobody conjugated to magnetic particles M-270 for analysis of very large proteins/complexes.

GFP VHH: anti-GFP Nanobody

Free sample

You can test the GFP-Trap yourself. Just request a free sample here:

FAQs

Can GFP antibodies detect YFP, CFP, or other derivatives?
That depends on the antibody. Most GFP antibodies also recognize other derivatives, especially if there are just a few amino acid substitutions compared to GFP or EGFP. For example, the GFP-Trap recognizes AcGFP, Clover, eGFP, Emerald, GFP, GFP5, GFP Envy, GFP, S65T, mGFP, mPhluorin, PA-GFP, Superfolder GFP, TagGFP, TagGFP2, monomeric eGFP K206A, CFP, YFP, Citrine, eCitrine, eYFP, Venus, Ypet, BFP.

Is GFP still fluorescent after fixation?
There is no general answer to this question as it depends on the fixation method and procedure.