GFP

Green Fluorescent Protein (GFP) is a natural protein first isolated from the jellyfish Aequorea victoria. The protein absorbs energy from blue light and in response emits a green glow. Since its first isolation in 1962, the protein and even more so the corresponding gene has transformed biomedical research. Martin Chalfie, Osamu Shimomura, and Roger Tsien received the Nobel Prize in chemistry for their discovery and development of GFP in 2008. The search for the perfect fluorescent protein (FP) is an ongoing endeavour and has led to the development of many GFP variants with different functional and spectral properties. A well-established variant, which facilitates the practical use of GFP in a variety of different organisms and cells contains a chromophore mutation and is called enhanced GFP or EGFP. Next to jellyfish derived GFP variants there are additional, non-related FPs like mNeonGreen and TurboGFP. ChromoTek offers affinity products for those products, too.

CRISPR/Cas

CRISPR/Cas9 mediated genetic engineering has emerged as a powerful method due to its relative ease of use. Hence, CRISPR/Cas9 has been widely adopted for the fluorescent protein tagging of endogenous genes. Corresponding GFP-fusion proteins are expressed at endogenous level and can be effectively immunoprecipitated using GFP-Trap® and detected using GFP-Booster where needed.

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Enhancing the immunofluorescence of GFP-tagged proteins

GFP and its derivatives may have low signal intensities depending on expression level and method of fixation of the sample for imaging. Some methods like HCl treatment for BrdU-detection, EdU-Click-iT™ treatment, or heat denaturation for FiSH may even lead to disruption of the GFP’s signal. ChromoTek’s GFP-Boosters is a GFP-Nanobody conjugated to fluorophores that specifically binds to GFP to recover the fluorescent signal.

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Immunoprecipitation and Co-Immunoprecipitation

Researcher may want to know both where their protein of interest is located inside a cell and what proteins it interacts with. Therefore, GFP and other FPs are commonly utilized as a protein tag for immunoprecipitation, too. ChromoTek’s GFP-Trap has a fair share of driving the use of GFP as a protein tag because of its ease of use and unprecedented performance. Using GFP as a tag has some advantages, because the same GFP-fusion protein construct can be used for microscopy, immunoprecipitation, and other biochemical assays. ChromoTek’s single domain GFP antibody coupled to agarose or magnetic agarose beads is called GFP-Trap or sometimes GFP-beads. GFP-Trap binds to GFP-fusion proteins with very high affinity and low background. By applying harsh washing conditions, background can be even further reduced.

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Mass spectrometry

Interacting partners of a protein, which have been isolated by Co-IP, can be identified by Mass Spectrometry (MS). GFP-Trap is particularly beneficial for MS analysis following Co-IP, because of its reliable performance, high affinity, and low background of protein contaminations. The superior stability of the GFP-Trap allows to apply stringent washing conditions. In most cases, even buffers containing chaotropic agents or detergents can be used, whereas these would inactivate other IgG-based or Streptavidin-based affinity resins. Therefore, background can be further reduced, which increases the sensitivity of the sample analysis. This may be required for applications like the analysis of some post-translational modifications (PTMs).
It is recommended to conduct sample preparation on-bead to avoid loss of sample by incomplete elution from the Nano-Trap. The small size of the GFP-Nanobody only adds 4-5 easy to identify peptides after tryptic digest.

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GFP as tag for membrane protein purification

The expression and purification of membrane proteins may be time-consuming and cumbersome. The use of GFP as a protein tag fused to a membrane protein can be advantageous as it facilitates direct monitoring and visualization of the process. The GFP moiety of the fusion protein might also be favorable for enhancing the solubility of the recombinant membrane protein. Because of the extraordinary stability of the GFP-Trap affinity resin, a variety of detergents in high concentrations can be used for purification. The 1 pM high affinity of GFP-Trap enables to effectively purify in large sample volumes when needed.

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GFP as tag for microscopy and protein purification

Green fluorescent protein (GFP) is the most widespread fluorescent reporter of gene expression, protein localization, and for protein interaction in cells, tissues, and organisms. For many reasons, is advantageous to utilize the GFP detection tag as a capture tag, too. However, capture applications, i.e. the purification of GFP-tagged proteins requires an affinity resin that binds with high yield and purity. ChromoTek’s GFP-Trap® was the first GFP affinity reagent that enables the effective purification of GFP fusion proteins without need of additional purification tags.

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Super-resolution microscopy

Super-resolution microscopy (SRM) describes a number of microscopy techniques that enable imaging of cellular structures at resolutions beyond the Abbe diffraction limit. This limit restricts the optical resolution in conventional light microscopy. Resolutions below the diffraction limit reveal more details of cellular structures, which allows a better understanding of many biological processes. The application of green fluorescent protein (GFP) in super-resolution microscopy techniques like 3D-SIM, STED, and STORM SRM may be compromised by insufficient photostability and quantum efficiency of GFP. Furthermore, GFP and its derivatives are less bright than fluorescent dyes. GFP-Boosters, i.e. ChromoTek’s fluorophore-conjugated anti-GFP Nanobody against common derivatives of GFP overcome these problems, when binding the GFP-fusion protein. The fluorescent signal thereby can be recovered and amplified.

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Looking for references? Please check our literature database.

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