AffiniPure-VHH® Anti-GFP antibody

Green Fluorescent Protein (GFP) is a commonly used protein tag for generating fusion proteins that can be expressed in various prokaryotic and eukaryotic systems. The GFP tag is a useful reporter molecule because it does not require exogenous substrates or cofactors to generate fluorescence. GFP is employed in numerous applications, including quantification of gene expression, protein localization within living organisms, studying protein interactions, and as a biosensor. The addition of an Anti-GFP antibody may be used to improve experimental performance and overcome limitations that use of the tag itself may incur.

About Jackson ImmunoResearch AffiniPure-VHH® Fragment Alpaca Anti-GFP Antibodies

Specificity for avGFP and its derivatives

Jackson ImmunoResearch AffiniPure-VHH® Anti-GFP is a polyclonal VHH fragment from an Alpaca host. It can be used to detect Aequorea victoria Green Fluorescent Protein (avGFP) and its derivatives, including EGFP, ECFP, and EYFP (Fig. 1).

Figure 1: We tested the reactivity of AffiniPure-VHH® Anti-GFP antibody to a range of fluorescent proteins by ELISA. Fluorescent proteins known to exhibit positive reactivity to AffiniPure-VHH® Anti-GFP antibody, such as EGFP, ECFP, and EYFP, were coated at lower concentrations (left graph) than fluorescent proteins we did not expect to be detected by AffiniPure-VHH® Anti-GFP antibody (right graph). Results show that even at very low concentrations, AffiniPure-VHH® Anti-GFP antibody recognizes avGFP variants, but does not react with TagRFP, mTFP, phiYFP, mKate, Turbo GFP, mKusabiraOrange, dTomato, and mCherry. HRP-AffiniPure® Goat Anti-Alpaca VHH antibody (128-035-232) was used as a detection antibody at a 1:20,000 dilution.

Advantages of the AffiniPure-VHH® format

Novel host and novel format

Camelid species such as Alpaca and Llama produce a unique class of antibodies composed only of heavy chains. The antigen-binding fragments (Fab) are also referred to as Variable Heavy-Chain only fragment antibodies (VHH Fragments), or NANOBODIES®. AffiniPure-VHH® Anti-GFP antibodies are comprised of this exciting, novel antibody format, which offers outstanding specificity and penetration, and are a fantastic solution for high-quality and high-resolution imaging.

Polyclonal Antibodies mean reliable and superior signal

Polyclonal detection reagents continue to offer greater sensitivity by amplifying signal, even from poorly expressing targets. Polyclonal antibodies provide a heterogeneous population of antibodies, each with a distinct paratope, thereby detecting a different portion of the GFP protein. By binding to sites across the entire protein of interest, rather than a limited specific site, region, or sequence, polyclonal AffiniPure-VHH® Anti-GFP antibodies can decorate the whole protein, enabling higher labeling efficiency and a brighter signal compared with a monoclonal nanobody* (Figure 2).

Figure 2: The polyclonal format of AffiniPure-VHH® antibodies enables sensitive detection of target antigens.

Versatile detection of the GFP protein across a range of applications

An Anti-GFP antibody can amplify the signal from GFP expressed by cells in IHC or IF experiments. Our AffiniPure-VHH® Anti-GFP antibodies may be used in ELISA, IHC, ICC, IP, Flow cytometry, and WB.

Imaging with AffiniPure-VHH® Fragment Alpaca Anti-GFP antibodies

Jackson ImmunoResearch AffiniPure-VHH® Anti-GFP antibodies are available conjugated to a range of Alexa Fluor® dyes. Designed for fluorescence microscopy, these bright dyes, combined with the polyclonal format of the AffiniPure-VHH® Anti-GFP antibodies, deliver bright target signal and spectacular signal amplification.

There are many uses for AffiniPure-VHH® Anti-GFP antibody conjugates, adding versatility to experiments or circumventing the limitations that the GFP tag can present.

Figure 3: AffiniPure-VHH® Anti-GFP antibody is available in a range of fluorophore options, enabling GFP tag detection to be switched to an alternate channel, avoiding autofluorescence, or to accommodate detection of multiple targets. Figure 3 shows HeLa cells transfected with EGFP-tubulin and stained with different fluorescent conjugates of AffiniPure-VHH® Anti-GFP antibody. Panel A, Alexa Fluor® 555 AffiniPure-VHH® Anti-GFP antibody (600-564-245), Panel B, Alexa Fluor® 594 AffiniPure-VHH® Anti-GFP antibody (600-584-245), Panel C, Alexa Fluor® 647 AffiniPure-VHH® Anti-GFP antibody (600-604-245). All figures were captured using a Nikon Ji wide-field microscope and Fusion camera, with corresponding excitation and emission filters for each fluorophore.

AffiniPure-VHH® Fragment Alpaca Anti-GFP antibody Conjugates for Immunohistochemistry

AffiniPure-VHH® Anti-GFP antibody is also available as a biotin conjugate, facilitating its use in chromogenic IHC staining techniques and amplification protocols such as ABC (Avidin-Biotin-Complex) and LSAB (Labeled Streptavidin-Biotin). Switching to a non-fluorescent detection system enables the use of traditional light microscopy techniques, such as chromogenic IHC.

Detection of GFP by flow cytometry using AffiniPure-VHH® Fragment Alpaca Anti-GFP antibodies: Case Study

Flow cytometry is a valuable technique for screening and quantifying protein expression, particularly when proteins are located on the cell surface. Flow cytometry frequently uses the GFP tag as a reporter molecule to track protein expression. Learn how Jackson ImmunoResearch AffiniPure-VHH® Fragment Alpaca Anti-GFP antibody conjugates can detect a GFP fusion protein expressed on yeast cells by flow cytometry.

References

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