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Properties of proteins: Difference between revisions
→Melting point
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== Melting point == | == Melting point == | ||
Thermofluor is a fluorescence-based thermal stability assay. | Thermofluor is a fluorescence-based thermal stability assay. From [http://www.uni-leipzig.de/~straeter/newsletter/images/letter_0310/thermofluor.pdf]: The experiment is easy to perform and requires no prior knowledge of the protein properties. Every standard real‐time PCR machine – available in many research labs – is suitable | ||
to perform this high‐throughput experiment in a short time (1‐2 hours). Only relatively little | |||
protein (500 µl of protein at 1‐2 mg/ml for a 96‐well experiment) and a dye (e.g. SYPRO® Orange, Invitrogen) are required. Proteins containing prosthetic groups (e.g. FAD) may not even require an external fluorophore if the intrinsic signal can be used during the Thermofluor experiment. For FAD‐containing proteins this method has been termed ThermoFAD. | |||
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Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N. (2009) Predicting melting temperature directly from protein sequences. Comput Biol Chem. 33, 445-450 at [http://dx.doi.org/10.1016/j.compbiolchem.2009.10.002]. | Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N. (2009) Predicting melting temperature directly from protein sequences. Comput Biol Chem. 33, 445-450 at [http://dx.doi.org/10.1016/j.compbiolchem.2009.10.002]. | ||
They have a list of 35 different proteins with their Tms with the references from where they obtained their data, and their Tm Index program is available at [http://tm.life.nthu.edu.tw/]. | They have a list of 35 different proteins with their Tms with the references from where they obtained their data, and their Tm Index program is available at [http://tm.life.nthu.edu.tw/]. | ||
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A specific case which shows that Tm does not need to be high: the protein with PDB-ID 1ofc had a melting temperature of 37°C (from CD), which was supported by the fact that it did not express in E.coli at that temperature. | A specific case which shows that Tm does not need to be high: the protein with PDB-ID 1ofc had a melting temperature of 37°C (from CD), which was supported by the fact that it did not express in E.coli at that temperature. | ||
At 20°C it expressed to about 60mg / (liter LB), could be concentrated to more than 100mg/ml, crystallised at room temperature and diffracted to 1.9A. The initial purification steps were done at 4°C. | At 20°C it expressed to about 60mg / (liter LB), could be concentrated to more than 100mg/ml, crystallised at room temperature and diffracted to 1.9A. The initial purification steps were done at 4°C. | ||