GFP fusion protein Technology - Proteomic Analysis tools Save time and reduce research costs

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• published: 25 Feb 2014
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Sandia Biotech http://wwww.SandiaBio.com This is a properly expressed protein with our exposed tagging fragment (the small, 16 aa, does not interfere with structure, conformation or function as whole GFP, other tags) The tagged protein self assembles with our heavier detection fragment ( this larger 27KD fragment binds only the small fragment in a very specific and irreversible manner) Resulting in a fluorescent GFP fusion protein It is important to note that neither fragment can fluoresce alone but only when bound to each other, therefore there are no false positives caused by non-specific signaling. As you are well aware, determining the level of expression, correct conformation and solubility of proteins is essential in drug discovery and development. This assay can provide structural information as a misfolded, aggregated or insoluble protein will not bind the detection fragment and as a result, not generate a signal. An additional advantage of this technology is that it can circumvent the steric limitations of antibodies with the relatively small size of the tagging and detection fragments. The tagging fragment, at a 1:1 molar ratio to ligand does not interfere with activity and allows for kinetic and functionality assays. Fragment complementation occurs within minutes and the interaction saturates in a matter of hours. The tag offers the same advantages as a radiometric assay without the hazards and can be used on standard fluorescent analysis equipment used for direct or immunofluorescent assay. The 1-10 detection reagent is an engineered fragment of GFP (CFP or YFP) that specifically and irreversibly binds the tagging fragment on the protein or peptide of interest. This very specific binding eliminates the need for antibody or aptamer detection. Because the system only fluoresces when it self-assembles to the tagging fragment, there is no non-specific signaling, as is the case with antibodies, is eliminated. Further, there is little or no need for washing while blocking and incubation steps are not required. The entire fluorescent protein complex is only 27Kd which is roughly 5.6 times smaller than IgG at 150Kd. This allows for more effective entry into permeablized cells and bind target proteins and peptides within a cell. And since the split detection system does not fluoresce unless both fragments have self assembled, no extra washing is necessary. The sample can be analyzed soon after staining. http://youtu.be/sJ5g-KJK3yc