Green fluorescent protein (GFP), GFP antibodies, and GFP assay kits

GFP antibodies are often used in western blotting to measure the amount of GFP-tagged proteins, in IHC and cell staining to visualize the localization of the GFP-tagged protein, where GFP’s own fluorescence is not sufficient, and in IP and ChIP to pull down complexes containing the GFP-tagged proteins.

GFP ELISA kits and other GFP assay kits are used to rapidly quantify the amount of GFP fluorescence, and the amount of GFP protein in a sample.

Learn below about how GFP is used as a protein tag, and how to choose our best GFP antibody and GFP assay kit for your research.

Product highlights

What is GFP?

GFP (Green fluorescent protein) is a naturally occurring fluorescent 27kDa cytoplasmic monomeric protein that was first isolated from the jellyfish Aequorea victoria. It emits green light (peak emission 509 nm) and is excited by blue to ultraviolet wavelengths (peak excitation 395 nm).

 
How is GFP used as a research tool?

GFP is non-toxic and stable in cells and compatible with many model organisms, making it a useful tool in molecular and cellular biology. When GFP-like ab84191 is introduced into cells, it can be used for:

• Fluorescent microscopy to study the localisation of GFP-tagged proteins. GFP antibodies are often also used to visualise the GFP; this is particularly useful for visualising low-abundance proteins, as the signal is amplified, or when using fixed samples where fluorescence may be quenched. Chicken GFP antibodies, such as ab13970, are very popular as they can be used with fluorescent-dye conjugated secondary antibodies, alongside rabbit and mouse primary antibodies to other proteins in multi-color imaging.

• Reporter assays where GFP is placed downstream of a specific promoter. Researchers can determine how and when a specific promoter is turned on by visualizing and quantifying GFP using microscopy or flow cytometry. The most cited GFP antibody on the market ab290 with over 3,000 publications, is frequently used in quantitative reporter assays.

• Signal transduction studies using FRET (fluorescence resonance energy transfer) to understand protein interactions in signaling pathways. GFP variants, eg, BFP and YFP (see below), are beneficial for FRET studies.

• As a biosensor, GFP can be modified so that its fluorescence changes in response to different cellular changes, eg, pH shifts, calcium flux and ATP levels. Quantifying GFP then allows researchers to correlate signal intensity with biological function. The amount of GFP signal can be quantified using a fluorescent plate reader when using a GFP quantification kit, ab235672. This allows for GFP quantification in liquid samples such as cell lysates, tissue extracts, cell culture supernatants, and bodily fluids such as serum. Alternatively, a GFP ELISA kit can be used, such as ab171581, where enzymatic detection is used with TMB as the colorimetric substrate.

In addition to GFP’s use in live cells, it is also frequently used for:

• Protein purification where anti-GFP antibodies are bound to a solid support and bind GFP-tagged proteins in the cell lysate.

• Isolating protein complexes by co-immunoprecipitation (co-IP), where a cell lysate containing GFP-tagged proteins is incubated with an anti-GFP antibody, like ab6556. The antibody–protein complex can then be isolated and analysed by western blot. GFP antibodies, particularly ab290 with over 500 citations in ChIP, are also commonly used in chromatin IP (ChIP) studies.

• Verifying fluorescent microscopy findings by using western blot to compare GFP protein concentrations under different experimental conditions.

Figure 1. GFP antibody ab290 staining TRF2-GFP fusion protein with green fluorescent dye-conjugated secondary antibody.

GFP variants

GFP has been genetically modified to create new variants such as eGFP, BFP, CFP, and YFP, which offer different advantages:

• eGFP (enhanced GFP) – This is the most widely used variant and was created by introducing two mutations (F64L, S65T). With excitation and emission wavelengths of 488 nm and 507 nm, respectively, it is 35x brighter than wild-type GFP.

• BFP (Blue Fluorescent Protein) –Introducing the mutation Y66H allows the emission of blue fluorescence by changing the excitation and emission wavelengths to 383 nm and 445 nm, respectively.

• CFP (Cyan Fluorescent Protein) –Introducing the mutation Y66W allows blue-green fluorescence emission by changing the excitation and emission wavelengths to 435 nm and 485 nm, respectively.

• YFP (Yellow Fluorescent Protein) – Introducing the mutation T203Y allows the emission of yellow fluorescence by changing the excitation and emission wavelengths to 514 nm and 527 nm, respectively.

The above mutants are all fluorescent proteins derived from A. Victoria, which can emit light in the blue to yellow range of the spectrum and allow for multi-colour labelling and FRET (fluorescence resonance energy transfer). Where red fluorescence is required, RFP (red fluorescent protein) derived from the coral Discosoma striata and variants are used instead of GFP. RFP antibodies, such as ab62341 and ab185921, are used similarly to GFP antibodies.

Which are the most popular GFP antibodies and GFP assay kits?

The two most highly cited Abcam GFP antibodies are rabbit polyclonal ab290, with over 3,000 publications, and chicken polyclonal ab13970, with over 3,000 publications.

The single-wash, 90-minute, colorimetric GFP ELISA kit ab171581 uses our SimpleStep ELISA® technology and is our most popular GFP assay kit. It is available in 96 well and 384 well options, and has a fluorescent ELISA version available – ab229403.
It is complemented by our no-wash GFP quantification kit ab235672, which is used with a fluorometric plate reader to quantify the amount of GFP fluorescence, and includes a GFP standard curve.

Figure 2. IHC analysis of GFP transgenic mouse pancreas using GFP rabbit monoclonal ab183734.

How to choose the best GFP antibody for your research

Our anti-GFP antibodies are known to recognize the variants of Aequorea victoria GFP, including eGFP, YFP, sfGFP, CFP, BFP, and others. They are used both as GFP tag antibodies and for the analysis of GFP alone.

For multi-color imaging when a chicken antibody is not suitable, we supply anti-GFP antibodies raised in goat, mouse, and rat.