ChromoBlog

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

Posted by Christoph Eckert on May 25, 2021 5:13:30 PM

1. What is GFP?

2. Origin of GFP

3. How does GFP work?

4. What are GFP-tagged proteins and GFP-fusion proteins

5. GFP emission spectrum and excitation peaks

6. Which applications can GFP be used for?

7. GFP derivatives

8. Structure of GFP

9. Why is GFP fluorescent?

10. Size of EGFP

11. Properties of EGFP

12. EGFP sequences

13. Anti-GFP antibodies and Nanobodies

14. GFP beads

15. Free sample

16. FAQs

 

1. 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.

 

2. 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.

 

3. 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.4. What are GFP-tagged proteins and GFP-fusion proteins.

 

4. 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.

 

5. GFP emission spectrum and excitation peaks

GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm.

 

Excitation max

Emission max

GFP

395 nm and 475 nm

509 nm

EGFP (most common derivative, see below)

488 nm

507 nm

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

 

6. 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)


7. 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.

 

8. 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.

EGFP structure & Fluorescent core: Chromophore structure

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

 

9. 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, actually a two-ring 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.

 

10. Size of EGFP

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

 

11. Properties of EGFP

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

 

12. 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

 

13. Anti-GFP antibodies and Nanobodies

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


14. 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

 

15. Free sample

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

GFP-Trap sample

16. 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 (click here for a complete list).

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

Topics: GFP, GFP VHH, GFP Nanobody, EGFP

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