GFP & EGFP: Properties, Spectrum & Detection Antibodies

발행일: 2026-06-24 조회수: 327

Green Fluorescent Protein (GFP) and its enhanced variant EGFP are the most widely used fluorescent reporter proteins in cell biology, enabling real-time visualization of gene expression, protein localization, and dynamic cellular processes. While GFP fluorescence can be observed directly, many experimental workflows require antibody-based detection of GFP/EGFP — particularly for Western blot confirmation, IHC on fixed tissue where fluorescence is lost, co-immunoprecipitation of GFP-tagged complexes, and signal amplification in low-expression systems.

This guide covers the key properties of GFP/EGFP, common variants and their cross-reactivity, and how to choose the right anti-GFP antibody format (conventional IgG vs. VHH nanobody) for your application.

GFP vs. EGFP: Key Differences

Property	Wild-Type GFP	EGFP
Source	Aequorea victoria jellyfish	Engineered from wild-type GFP
Key mutations	None (wild-type)	F64L + S65T (improved folding and brightness)
Excitation / Emission	395/509 nm (major) and 475/509 nm (minor)	488/507 nm (single peak, optimized for standard laser lines)
Brightness	Moderate	~35× brighter than wild-type at 488 nm excitation
Maturation at 37°C	Slow and incomplete	Fast and efficient
Molecular weight	~27 kDa	~27 kDa
Codon optimization	Jellyfish codons (poor mammalian expression)	Human codon-optimized

Practical implication: EGFP is the standard in modern research. If you see "GFP" in a commercial vector or published protocol from the last ~15 years, it almost always means EGFP (or another enhanced variant), not wild-type GFP.

Common GFP Variants and Cross-Reactivity

Dozens of GFP variants exist with different spectral properties, but they share >95% sequence identity with EGFP. Most anti-GFP antibodies cross-react with all common variants. The table below shows which variants a typical anti-GFP antibody will detect:

Variant	Excitation/Emission	Color	Detected by Anti-GFP?
EGFP	488/507 nm	Green	Yes
EYFP / Venus / Citrine	514/527 nm	Yellow	Yes
ECFP / Cerulean / mTurquoise	433/475 nm	Cyan	Yes
EBFP / EBFP2	383/448 nm	Blue	Yes
mNeonGreen	506/517 nm	Green	No — derived from Branchiostoma, not Aequorea
mCherry / mRFP / tdTomato	Various (red)	Red	No — derived from Discosoma DsRed

Key rule: Anti-GFP antibodies detect Aequorea-derived variants (EGFP, EYFP, ECFP, EBFP and their derivatives) but do not detect non-Aequorea fluorescent proteins (mNeonGreen, mCherry, mRFP, tdTomato). For these, you need anti-RFP or anti-mCherry specific antibodies.

Why Use an Anti-GFP Antibody Instead of Direct Fluorescence?

Scenario	Why Antibody Detection Is Needed
Western blot	GFP fluorescence is destroyed by SDS denaturation and boiling. Anti-GFP antibody detects the denatured protein on the membrane.
IHC on FFPE tissue	Formalin fixation and paraffin embedding destroy or severely reduce GFP fluorescence. Anti-GFP + DAB chromogenic detection restores visualization.
Signal amplification	Weak GFP expression can be amplified using anti-GFP primary + fluorescent secondary for IF imaging.
Co-immunoprecipitation	Pull down GFP-tagged bait protein + interacting partners using anti-GFP antibody coupled to beads.
Super-resolution microscopy	Anti-GFP nanobodies (~15 kDa) provide smaller linkage error than conventional IgG (~150 kDa), improving localization precision in STORM/PALM.

Choosing the Right Anti-GFP Antibody

Format	Advantages	Best For
VHH Nanobody (~15 kDa)	10× smaller than IgG; penetrates dense structures; lower linkage error; single-domain stability; recombinant production	Super-resolution (STORM/PALM); live-cell nanobody traps (GFP-Trap); ChIP; samples with limited accessibility
Recombinant Monoclonal	Defined sequence; lot-to-lot consistency; single epitope; low background	WB, IF, flow cytometry; standardized protocols; multiplex
Polyclonal IgG	Multiple epitopes; tolerates partial denaturation; strong signal	WB (denatured GFP); IHC on heavily fixed tissue; IP where maximum capture is needed
HRP-Conjugated	Direct detection; no secondary antibody needed; faster protocol	WB (direct chemiluminescent detection); high-throughput screening

Anti-GFP/EGFP Antibodies from abinScience

Anti-GFP VHH Nanobody (SAA1151) — Cat# ZP144013 | WB, IF, FC, ELISA | 15 kDa, ideal for super-resolution

Anti-EGFP Monoclonal (1A190) — Cat# YP140025 | WB, IF, ELISA | Also in HRP

Anti-GFP Tag Recombinant (N86/38.1) — Cat# YP144013 | WB, IF, IHC, IP

Browse All 13 Anti-GFP/EGFP Antibodies →

abinScience Anti-GFP/EGFP Antibody Catalog

VHH Nanobodies

Product	Cat#	Clone	Applications
Anti-GFP VHH, Recombinant Nanobody	ZP144013	SAA1151	WB, IF, FCM, ELISA
Anti-GFP VHH, Recombinant Nanobody	ZP144023	SAA1152	WB, IF, FCM, ELISA
Anti-GFP VHH, Recombinant Nanobody	ZP144033	SAA1153	WB, IF, FCM, ELISA
Anti-GFP VHH, Recombinant Nanobody	ZP144043	SAA1154	WB, IF, FCM, ELISA
Anti-GFP VHH, Recombinant Nanobody	ZP144053	SAA1155	WB, IF, FCM, ELISA

Recombinant Monoclonal & Polyclonal Antibodies

Product	Cat#	Clone	Applications
Anti-GFP Tag Recombinant Antibody	YP144013	N86/38.1	WB, IF, IHC, IP
Anti-GFP Tag Recombinant Antibody	YP144023	SAA0301	WB, IF, IHC, ELISA, IP
Anti-EGFP Monoclonal Antibody (1A190)	YP140025	1A190	WB, IF, ELISA
Anti-EGFP Monoclonal Antibody (1A190), HRP	YP140925	1A190	WB, IF, ELISA
Anti-GFP Monoclonal Antibody (3H9)	YP144035	3H9	WB, IF, IP
Anti-GFP Monoclonal Antibody (5D7)	YP144045	5D7	WB, IF, IP
Anti-GFP Polyclonal Antibody	YP144014	—	WB, IF, IP, ELISA
Anti-GFP Polyclonal Antibody	YP144024	—	WB, IF, IP, ELISA

Frequently Asked Questions

Q: Will an anti-GFP antibody detect my mNeonGreen or mCherry construct?

No. Anti-GFP antibodies only detect proteins derived from Aequorea victoria GFP (EGFP, EYFP, ECFP, EBFP, and their monomeric variants). mNeonGreen is from Branchiostoma lanceolatum, and mCherry/tdTomato are from Discosoma DsRed. You need species-specific antibodies for these proteins.

Q: What is a GFP-Trap and when should I use one?

A GFP-Trap is a VHH nanobody coupled to agarose or magnetic beads, used for fast, efficient immunoprecipitation of GFP-tagged proteins and their binding partners. It outperforms conventional anti-GFP IgG + Protein A/G bead IP because: (1) the nanobody binds with very high affinity (sub-nanomolar), (2) the small size reduces non-specific co-precipitation, and (3) the covalently coupled format avoids antibody heavy/light chain contamination on the eluted gel. abinScience Anti-GFP VHH nanobody (Cat# ZP144013) can be coupled to your own beads for custom IP applications.

Q: Can I use the same anti-GFP antibody for both WB and IF?

Yes, many anti-GFP antibodies are validated for both. On WB, the antibody detects denatured GFP (~27 kDa band). For IF, the same antibody detects native GFP in fixed cells, amplifying the fluorescent signal. Use an unconjugated anti-GFP primary + fluorescent secondary antibody for IF. For direct WB detection without secondary, use the HRP-conjugated format (e.g., abinScience Cat# YP140925).

Q: Why do I see a band higher than 27 kDa when blotting for GFP?

The expected band for GFP/EGFP alone is ~27 kDa. If your band is higher, it reflects the molecular weight of your fusion protein (target protein + GFP tag). For example, if your target protein is 50 kDa, the GFP-tagged fusion will appear at ~77 kDa. Always calculate: fusion MW = target MW + 27 kDa (GFP). If you see an unexpected 27 kDa band in addition to the fusion band, it may indicate cleavage of the GFP tag from the fusion protein.

References

1. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-544. doi: 10.1146/annurev.biochem.67.1.509

2. Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1):33-38. doi: 10.1016/0378-1119(95)00685-0

3. Ries J, Kaplan C, Platonova E, et al. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods. 2012;9(6):582-584. doi: 10.1038/nmeth.1991

GFP Nanobodies + Monoclonals + Polyclonals

13 anti-GFP/EGFP antibodies including 5 VHH nanobody clones. Unconjugated and HRP formats for WB, IF, IHC, IP, and ELISA.

Shop Anti-GFP Antibodies →

All products are for research use only (RUO). Not intended for diagnostic or therapeutic use. For technical support, contact info@abinscience.com.