CoralHue® Fluo-chase Kit
Protein-Protein Interaction Detection System
- Protein-protein interaction (PPI) analysis
- Direct visualization of PPIs in living cells
- Easy construction
CoralHue® Fluo-chase Kit is based on the protein fragment complementation assay using our
proprietary fluorescent protein , CoralHue® monomeric Kusabira Green (mKG).
mKG is split into two inactive fragments, mKG_N and mKG_C, and do not emit
fluorescence on their own.
With the CoralHue® Fluo-chase Kit the protein of interest (A) is expressed as a fusion protein
to the mKG_N (A-mKG_N) and the mKG_C is fused to the second protein of interest (B),
(B-mKG_C).
Both vectors encoding mKG fragment fusion protein are co-introduced into mammalian cells. When the mKG fragments are in close proximity due to the interaction of A and B, the mKG fragments form a beta-barrel structure and emit green fluorescence.
Non fluorescent fragments
mKG_N and mKG_C (left panel) or p65-mKG_N and p50-mKG_C (right panel) were co-expressed in HEK293T cells. After a 24h incubation, cells were imaged on an inverted microscope. HEK293T cells expressing the N terminal fragment of mKG (mKG_N) and/or the C terminal fragment of mKG (mKG_C) absolutely had no fluorescent signal.(p65-mKG_N ; 190 to 291 amino acid sequence of p65(RELA) fused to mKG_N. p50-mKG_C ; 247 to 352 amino acid sequence of p50(NFKB1) fused to mKG_C.)
There is the possibility of a nonspecific mKG fluorescent signal from an unexpected protein-protein interaction. For example, in our original experiments, HEK293T cells expressing both the mKG_N with a flexible linker and the p65-mKG_C emitted a nonspecific fluorescent signal. The intensity of the signal was about 20% of the p65-mKG_N and p50-mKG_C co-expressing HEK293T cells. Why the unexpected interaction occurred is now under evaluation.
Easy construction
For a topological variety of mKG fragments and proteins of interest, CoralHue® Fluo-chase Kit contains four regioselective vectors.Using the vector sets, all eight possible patterns for the proteins of interest (A and B) can be examined to select the most favorable fusion construct.
The lower panel shows all possible patterns between the cell cycle-related factors of p25 and Cdk5 interaction. Two of the eight pairs (mKG_N-Cdk5 + mKG_C-p25 and mKG_N-Cdk5 + p25-mKG_C ) successfully emitted bright fluorescent signals however, two other pairs (mKG_C-Cdk5 + mKG_N-p25 and mKG_C-Cdk5 + p25-mKG_N ) failed to emit a fluorescent signal.
Detection of PPIs using CoralHue® Fluo-chase Kit largely depends on the property of the protein complex, such as molecular mass, the direction of the N and C terminals, molecular proximity to each terminal, linker sequence, and so on. This product does not ensure the ability to detect all PPIs in nature.
Fluorescent properties of monomeric Kusabira Green (mKG)
Download mKG spectra Ex, Em (txt.)
| CHARACTERISTIC | Kusabira-Green |
|---|---|
| Fluorescence color | Green |
| Oligomerization | monomer |
| Excitation max (nm) | 494 |
| Emission max (nm) | 506 |
| Molar extinction coefficient (M-1cm-1) | 63,200 (494 nm) |
| Fluorescence quantum yield | 0.57 |
| Brightness*1 | 36.0 |
| pH sensitivity | pKa=6.1 |
| Molecular weight (kDa) | 24.5 |
*1Brightness: Molar extinction coefficient x Fluorescence quantum yield /1000.
The CoralHue® monomeric Kusabira Green(mKG) was co-developed by Amalgaam and the Laboratory for Cell Function and Dynamics, the Advanced Technology Development Center, the Brain Science Institute, RIKEN and Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC). This work was supported by a grant from the New Energyand Industrial Technology Development Organization (NEDO) of Japan.
Use of this product by for-profit organizations, please contact us at info@amalgaam.co.jp
Recommended antibodies
CoralHue® mKG_N and mKG_C can be recognized using antibodies as shown below.
- Anti-monomeric Kusabira-Green N-terminal fragment, Code No.M148-3
- Anti-monomeric Kusabira-Green C-terminal fragment, Code No. M149-3
References
1:Sequential binding of cytosolic Phox complex to phagosomes through regulated adaptor proteins: evaluation using the novel monomeric Kusabira-Green System and live imaging of phagocytosis.
Ueyama T, Kusakabe T, Karasawa S, Kawasaki T, Shimizu A, Son J, Leto TL, Miyawaki A, Saito N.
J Immunol. (2008) 181:629-40.
2:Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.
Hashimoto J, Watanabe T, Seki T, Karasawa S, Izumikawa M, Seki T, Iemura S, Natsume T, Nomura N, Goshima N, Miyawaki A, Takagi M, Shin-Ya K.
J Biomol Screen. (2009) 14: 970-979
