The OptiTherm kit offers a systematic solution design and fluorescence-based stability assay for thermal stabilization of protein samples.
Some protein samples clump together and aggregate when they expose hydrophobic patches. The target-specific choice of buffer type, pH, salt, organic and additive can reduce this behavior. This OptiTherm Protein Thermal Stability Kit helps to identify the solution parameters that provide highest thermal stability.
Use the OptiTherm Protein Thermal Stability Kit to:
- Identify the best buffer, pH, salts and additives for protein production, storage and analysis
- Analyze effects of protein sequence changes on stability
- Compare protein batches (quality control)
One OptiTherm kit contains consumable materials to assay solution conditions for up to six (6) different protein samples.
The OptiTherm Thermal Stability Kit is designed to enhance the thermal stability of protein samples. It is best used when stability is a known problem.
- Identifying the best buffer, pH, salts and additives for protein production, storage and analysis
- Analyzing effects of protein sequence changes on stability
- Comparing protein batches (quality control)
Some protein samples clump together and aggregate when they expose hydrophobic patches. The target-specific choice of buffer type, pH, salt, organic and additive can reduce this behavior. This OptiTherm Protein Thermal Stability Kit helps identifying the solution parameters that provide highest thermal stability.
This is how OptiTherm works
The OptiThermKit contains a systematically varied array of formulations and TFluor Dye. The user’s protein sample is mixed with the dye and formulations and then inserted into a temperature scanning fluorescence plate reader. A temperature scan is run while the TFluor fluorescence is recorded. Data is analyzed to identify formulation that renders the protein most temperature stable.
Background: The thermal stability of the protein preparation depends on the solvent properties. Hence, changes in melting temperatures are noted when the solvent varies with respect to pH, type and concentration of salts, organics and additives. Typically, proteins become more flexible and expose hydrophobic patches with increasing temperature. This thermal unfolding can be monitored with the TFluor™ Dye, an environmentally sensitive reporter molecule that increases its fluorescence when it binds to hydrophobic protein patches.
PROTOCOL: IDENTIFY CONDITIONS THAT THERMALLY STABILIZE PROTEIN SAMPLES:
- Dilute supplied TFluor™ Dye and pipette into side of Q-PCR plate well.
- Pipette protein sample to the opposite side of the well.
- Add 25 uL of formulations from the OptiTherm Formulation Plate.
- Seal the Q-PCR plate, spin and insert plate into temperature scanning fluorescence plate reader. Run temperature scan while recording TFluor fluorescence.
- Analyze resulting fluorescence / temperature data and record the midpoint of thermal denaturation for each formulation. Compare all such characteristic thermal denaturation points and identify formulation that render protein most temperature stable.
Variation: include known or putative small molecule ligands or co-factors (ATP, NAD/H, Zn2+ etc.) to protein buffer prior to analysis with the OptiTherm kit.
The data of the OptiTherm experiments is analyzed using a temperature scanning fluorescence plate reader.
The sealed Q-PCR plate containing the sample, reagents and dye is inserted into the plate reader and a temperature scan is run while recording TFluor fluorescence.
The resulting fluorescence/temperature data is analyzed and the midpoint of the thermal denaturation for each formulation recorded. All such characteristic thermal denaturation points are compared and the formulation that render protein most temperature stable is identifies.
Consumables and Equipment required:
- Q-PCR plate (preferably white) with plate seal
- A temperature scanning fluorescence plate reader such as a Q-PCR instrument (BioRad Opticon™, Stratagene Mx4000, Roche LightCycler® 480 quantitative PCR BioRad Opticon™ is a trademark of Bio-Rad Laboratories, Inc. Stratagene Mx4000™ is a trademark of Life Technologies Corporation. Roche LightCycler® is a trademark of is a trademark of Roche Diagnostics Corporation)
Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P.(2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem. 2006 Oct 15;357(2):289-98.
Crowther GJ, He P, Rodenbough PP, Thomas AP, Kovzun KV, Leibly DJ, Bhandari J, Castaneda LJ, Hol WG, Gelb MH, Napuli AJ, Van Voorhis WC. Use of thermal melt curves to assess the quality of enzyme preparations. Anal Biochem. 2010 Apr 15;399(2):268-75. (2009)
Lavinder JJ, Hari SB, Sullivan BJ, Magliery TJ, High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J Am Chem Soc (2009) 131:3794-3795
Senisterra GA, Finerty PJ, High throughput methods of assessing protein stability and aggregation. Mol Biosyst (2009) 5:217-223
Charvériat M, Reboul M, Wang Q, Picoli C, Lenuzza N, Montagnac A, Nhiri N, Jacquet E, Guéritte F, Lallemand JY, Deslys JP, Mouthon F, New inhibitors of prion replication that target the amyloid precursor. J Gen Virol (2009) 90:1294-1301
Frequently Asked Questions:
What is the composition of the OptiTherm screens?
The OptiTherm reagents consist of a combinatorial solution set with a wide variety of pH, buffer types, charged & neutral organic additives and antioxidants. A detailed list of all the components are listed in the OptiResc brochure that is included Kit.
Are the OptiTherm buffers the same as in the OptiSol and OptiResc?
Yes, the buffers are the same.
Are the buffer concentrations listed in the data sheet final or the stocks?
The concentrations listed are those of the reagents in the wells of the OptiTherm Reagent Plate.
What is Solubilisin II?
Solubilisin II is a proprietary solution that has been shown to promote the solubility and protect proteins from aggregation over a wide range of pH values.
Can Solubilisin II used in PK studies in vitro?
Solubilisin II has not been tested for in vivo studies and should be used for in vitro studies only. Some of the reagents supplied with the OptiTherm kits may contain low amounts of sodium azide and are hence incompatible with live cells. The OptiTherm kit is for in vitro research use only, not for cell-based or animal research.
Do you have an MSDS form
Yes. Contact us for more information: firstname.lastname@example.org
For research use only.