"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio UniversityRating: 5.0
Green fluorescent protein (GFP) was isolated from the bioluminescent jellyfish Aequorea victoria by Osama Shimomura in 1962. It wasn't until 1994 that Martin Chalfie first applied it to monitor gene expression. The utility of GFP as a tool soon became apparent, and a raft of other useful fluorescent proteins in a rainbow of colors were isolated and developed, the value of GFP was later recognized in the 2008 Nobel Prize. Fluorescent proteins are invaluable in many aspects of life science, their fusions/chimeras enabling the detection and quantification of protein/gene expression, and the characterization of molecular interactions through FRET and other optical experiments. Jackson ImmunoResearch now offers polyclonal Rabbit Anti-GFP antibody conjugates for the detection of GFP and EGFP in various applications.
GFP is a useful expression reporter molecule because it does not require exogenous substrates or cofactors to generate fluorescence (Chalfie et al 1994). GFP expression can be employed to monitor and quantify gene expression and characterize protein localization in living organisms within the complex environment of the cell (Lippincott-Schwarz 2001), and may also be employed in biosensors (Chalfie et al., 1994, Stretton et al.,1997, Tsien 1998). Fusion proteins coding for GFP may be transiently transfected, or stable cell lines that express the fusion may be generated. GFP can be expressed in many expression systems, including bacterial, yeast, insect, and mammalian.
Developed by the laboratories of Thastrup and Falkow in the late nineties, enhanced GFP (EGFP) possesses a single point mutation (F64L) that improves the brightness and subsequent tractability of the molecule. EGFP is the most prevalent variant of GFP used in molecular biology currently. Other variants of the initial GFP have subsequently been developed that have different spectral characteristics giving rise to yellow and blue (cyan) fluorescent proteins.
After Shimomura's discovery of GFP scientists began to investigate the properties of fluorescent proteins from other organisms. These genetically encoded fluorescence reporters were also subject to modification to augment their spectral characteristics so that they would excite and emit at different wavelengths enabling their application in experiments requiring multicolor detection.
Anti-GFP antibodies allow the detection of the GFP protein when direct fluorescence detection is not possible, such as overlapping excitation or emission profiles in the presence of autofluorescence, when another reporter molecule is required (Western blotting), or when the fluorescence of the native GFP is compromised or not sufficient and needs to be amplified.
Jackson ImmunoResearch AffiniPure™ Rabbit Anti-GFP is an affinity-purified polyclonal antibody raised in rabbit and can be used to detect Aequorea victoria Green Fluorescent Protein (avGFP) and its derivatives, including EGFP, ECFP, and EYFP. The antibody is suitable for use in a number of applications and is available unconjugated or conjugated to reporter enzymes, Biotin, R-PE, or Alexa Fluor® dyes.
| Conjugate | Excitation Peak (nm) | Emission Peak (nm) |
|---|---|---|
| Alexa Fluor® 488 | 493 | 519 |
| Alexa Fluor® 555 | 552 | 572 |
| R-Phycoerythrin, R-PE | many, 488 | 580 |
| Alexa Fluor® 568 | 577 | 602 |
| Alexa Fluor® 594 | 591 | 614 |
| Alexa Fluor® 647 | 651 | 667 |
| Alexa Fluor® 680 | 684 | 702 |
| Alexa Fluor® 790 | 792 | 803 |
Table 2: Anti-GFP antibody conjugates
