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Fluorescent Protein Hub
High-sensitivity detection, amplification, and isolation reagents for the entire fluorescent protein spectrum.
1. Overview: The Biochemical Challenge
Fluorescent proteins (FPs) have revolutionized molecular biology by allowing researchers to track cellular dynamics, gene expression, and protein localization in living systems in real-time. However, a significant experimental bottleneck occurs when transitioning from live-cell observation to downstream biochemical analysis.
Native fluorescence relies entirely on the structural integrity of the protein's 11-beta-sheet barrel, which shields the internal chromophore. When this protective barrel is quenched by chemical fixation, destroyed by tissue clearing, or denatured during cell lysis for a Western Blot, the intrinsic fluorescence vanishes completely.
The Solution: Biorbyt provides a comprehensive, literature-validated portfolio of primary antibodies and nanobodies engineered specifically to detect both the folded (native) and denatured (linear) peptide sequences of these fluorescent fusion proteins, bridging the gap between live-cell imaging and biochemical validation.
2. Core Fluorescent Protein Families
Choosing the correct fluorescent reporter dictates your downstream detection strategy. Below are the major families of FPs and their corresponding Biorbyt product hubs.
The Green & Cyan Proteins
Wild-type GFP and its variants originated from the Aequorea victoria jellyfish. These have been heavily engineered for mammalian expression and rapid folding. Because their peptide sequences are nearly identical, Biorbyt’s highly specific anti-GFP antibodies will universally cross-react across this family.
- GFP / EGFP: The gold standard for live-cell imaging and transfection monitoring.
- Cyan & Yellow Frequently utilized in Fluorescence Resonance Energy Transfer (FRET) experiments.
- mNeonGreen: A uniquely bright green monomer from the lancelet Branchiostoma lanceolatum (requires specialized antibodies distinct from Aequorea-derived GFP).
The Red & Far-Red Proteins
Derived largely from Discosoma coral, red FPs penetrate mammalian tissue significantly better than green light, avoiding natural cellular autofluorescence.
- mCherry: A highly stable, monomeric workhorse optimized for direct fusion to target proteins without causing artificial aggregation.
- tdTomato: A tandem-dimer linking two chromophores. It is exceptionally bright and photostable, making it the premier choice for whole-animal lineage tracing and deep-tissue xenograft imaging.
- mScarlet: The newest generation of monomeric red FPs. Known for an exceptionally high quantum yield (~70%), it is ideal for low-expression systems and CRISPR knock-ins.
- Far-Red (mPlum, mKate2, E2-Crimson) Deep tissue penetrators used for in vivo imaging of sensitive stem cell populations.
3. Antibodies By Target & Color
Below is a comprehensive selection of our primary antibodies. We have meticulously curated this list to represent the full spectrum of fluorescent proteins, offering diverse host species to prevent cross-reactivity in your multiplexing panels.
Target Spectrum | Product Name / SKU | Applications | Size | Price |
|---|---|---|---|---|
Blue | BFP Goat Polyclonal Antibody | WB, IP, IF | 100 μg | $300 |
Cyan | CFP Goat Polyclonal Antibody | WB, IF, IHC-P | 100 μg | $300 |
Green | GFP Goat Polyclonal Antibody | WB, IF, IHC, IEM | 100 μg | $280 |
Orange-Red | tdTomato Goat Polyclonal Antibody | FACS, IF, IHC-P, WB | 100 μg | $280 |
Red | mCherry Goat Polyclonal Antibody | IF, IHC-Fr, WB, IEM | 100 μg | $280 |
Bright Red | mScarlet Goat Polyclonal Antibody | IF, WB, IEM | 100 μg | $300 |
Deep Far-Red | mPlum Goat Polyclonal Antibody | WB, IHC-P | 100 μl | $300 |
4. Applications & Validation Workflows
1
Immunohistochemistry (IHC) & Immunofluorescence (IF)
Spatial biology requires robust reagents that survive harsh antigen retrieval protocols. Biorbyt's polyclonal antibodies provide superior signal amplification in fixed tissue sections (FFPE or cryosections) by binding multiple distinct epitopes on the denatured fluorescent protein.
2
Western Blotting (WB)
To accurately quantify the expression of your fusion proteins, you must use primary antibodies that recognize the fully denatured linear sequence of the protein. Highly stable FPs like tdTomato require fully reducing lysis buffers and rigorous boiling prior to electrophoresis to ensure the beta-barrel unfolds completely for antibody access.
3
Immunoprecipitation (IP) & Co-IP
Isolating target protein complexes with traditional full-length antibodies results in heavy (~50 kDa) and light (~25 kDa) chain contamination on your subsequent Western blots. Biorbyt offers specialized single-domain Nanobodies (VHH) targeting FPs that eliminate this background interference entirely.
5. Frequently Asked Questions (FAQ)
Why do my RFP, mCherry, and tdTomato antibodies sometimes cross-react?
Because tdTomato, mCherry, and mPlum were all originally engineered from the same parent protein (DsRed from the Discosoma coral), they share high sequence homology. Polyclonal antibodies often recognize conserved linear epitopes across these variants.
Can I use a GFP antibody to detect EGFP, CFP, or YFP?
Yes. Enhanced GFP (EGFP), Cyan Fluorescent Protein (CFP), and Yellow Fluorescent Protein (YFP) share over 95% sequence identity with wild-type GFP. Most standard polyclonal GFP antibodies (like orb11604) will robustly detect all of these variants on a Western blot or in fixed tissue.
Why is tdTomato so much brighter than mCherry, and when should I avoid it?
tdTomato is a "tandem dimer" linking two chromophores together, giving it exceptional brightness and preventing the aggregation issues seen in older tetrameric reds. However, at ~54 kDa, it is twice the size of a standard 27 kDa monomeric FP. You should avoid tdTomato if you are directly fusing it to a small or sterically sensitive protein, as the large tag might disrupt the target protein's folding or subcellular localization. For direct fusions, monomers like mCherry, mScarlet, or EGFP are vastly superior.
My Western Blot shows no band for my fluorescent protein, but the live cells were glowing. What happened?
If the cells were glowing, the protein is present and properly folded. The issue is likely your antibody. Some antibodies only recognize the 3D conformational structure of the native 11-beta-sheet barrel. Once you boil your sample in SDS (denaturing the barrel), those conformational antibodies can no longer bind. Ensure you are using a primary antibody explicitly validated for Western Blotting, which guarantees it binds to the fully denatured linear peptide sequence.
6. Multiplexing & Co-Staining Strategies
Designing a complex multiplex assay requires careful consideration of host species to avoid cross-reactivity during secondary antibody incubation.
- Species Separation: If you are co-staining your mCherry reporter alongside a neural marker like NeuN (typically raised in Rabbit), utilize a Goat anti-mCherry primary antibody. This ensures your anti-Goat and anti-Rabbit secondary fluorophores do not cross-react and create false-positive overlaps.
- Direct Conjugates: To bypass secondary antibodies entirely, Biorbyt offers primary antibodies conjugated directly to fluorophores (e.g., FITC, HRP, or Biotin) to streamline your protocol.
7. Companion Bioreagents & Tools
To support your complete experimental pipeline, Biorbyt offers a comprehensive suite of reagents perfectly paired with our fluorescent protein portfolio.
Nanobody GFP VHH Single-Domain Antibody Alpaca-derived binder for ultra-clean Immunoprecipitation and Co-IP workflows without heavy/light chain background. Secondary Antibody Goat Anti-Rabbit IgG (HRP) Highly cross-adsorbed secondary antibody, optimized for extremely low background in IHC and Western Blot workflows. Custom Services Custom Conjugation Service Need a specific dye? Our scientific team can custom-conjugate any of our primary FP antibodies to your exact specifications.
8. Scientific Bibliography & Validation Sources
- Tsien, R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544.
- Shaner, N. C., et al. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology, 22(12), 1567–1572.
- Bindels, D. S., et al. (2017). mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods, 14(1), 53–56.
- Chudakov, D. M., et al. (2010). Fluorescent proteins and their applications in imaging living cells and tissues. Physiological Reviews, 90(3), 1103–1163.