Solubility and stability are important intrinsic properties of biological products, including proteins. Many proteins and peptides are poorly soluble, especially membrane proteins, which are not traditionally soluble at all. Many in vitro proteins require an environment in which the protein can stably retain its native conformation, otherwise the non-native aggregated structure of the protein may lose its normal function.
Understanding protein stability and solubility is essential for optimizing protein expression, purification, formulation, storage, and structural studies. Solubility and stability of proteins are universally desired in a wide range of applications such as general biochemical research, protein preparation in diagnostics and pharmaceuticals, and structural biology. A range of chemical factors affect the solubility and stability of proteins, including pH, buffer type, ionic strength, and specific additives. Preparation of high concentration, soluble and stable protein samples is often a difficult task, because proteins are often aggregated, precipitated, or denatured by their own and external influences.
Solubility and stability screening are designed to identify solution conditions that promote protein solubility and stability and minimize protein precipitation. Amerigo Scientific offers screening kits for rapid and simple identification of formulations that dissolve protein aggregates or maximize the thermal stability of protein samples. These kits are in 96- well format and can be used to compare relative solubility across common formulations with controls.
OptiSol™ kits and OptiPharma™ kits provide mixing, filtration and collection plates for elution measurement by A280 or BCA assay on a plate reader. The OptiSol™ kit includes a higher dynamic range of formulations for more aggressive solubilization. The OptiPharma™ kit includes only FDA-approved excipients that are safe for drug development. The formulations in OptiPharma™ are those found to be most beneficial for increased solubility, based on more than a decade of formulation experience of pharmaceutical companies.
|OptiPharma - Protein Solubility Screening Kit||I - IV||1 pack; 3 packs|
|OptiSol - Protein Solubility Screening Kit||I - IV||1 pack; 3 packs|
OptiSol™ and OptiPharma™ protein solubility screening kits are suitable for a wide diversity of protein sizes, from a few kDa to 250 kDa. The filter plates should be selected based on the molecular weight of the dissolved protein molecules.
|I||Peptides, size < 10 kDa|
|II||Small protein molecules > 10 kDa and < 25 kDa|
|III||Protein molecules up to 90 kDa|
|IV||Large protein molecules (including antibodies and oligomers) up to 250 kDa|
Using the principle that soluble protein can pass through the filter but aggregated proteins cannot, the wells containing soluble proteins are filtered to obtain desired proteins, while wells containing aggregates are filtered to obtain no proteins. Depending on the information required and the nature of the starting sample, the protein solubilization screening kits can be used to determine the conditions that maintain protein solubility when a certain stress is applied, or to solubilize protein aggregates.
Figure 1. Protein Solubility Profiling
Figure 2. Solubilizing Aggregated Protein
This undesirable process of protein aggregation is usually caused by increased temperature, intense agitation, addition of ligands or molecular binding partners, or simply storage of samples for a period of time. Protein aggregation can be avoided by proper selection of pH, salt, or stabilizing additives. OptiSol™ and OptiPharma™ protein solubilization screening kits contain a series of systematically different buffers (pH 3 to 10 for OptiSol™ and pH 3 to 8.5 for OptiPharma™) and a series of solubility enhancers (salts, amino acids, sugars, polyols, and reducing agents). A total of 90 or 93 different formulations with solubility enhancers can be tested in a single, label-free experiment. The remaining 6 or 3 wells are for control experiments.
The solution design and array-based filtration technology of OptiSol™ and OptiPharma™ systems enables to identify formulations that protect target proteins from aggregation or gently solubilize aggregated protein samples. The accompanying Protein Dashboard™ helps spot protein behavior trends and identify key solvent factors for optimal protein solubilization within a single experiment.
OptiTherm™ kits include the formulations for more aggressive solubilization found in the OptiSol kits. The kits are based on fluorescence denaturation assay of thermal stability which can be measured using real-time PCR (quantitative PCR, qPCR) instruments. Fluorescence profiles across a temperature profile are used to identify the relative unfolding temperatures of the protein in each formulation.
|OptiTherm - Protein Solubility Screening Kit||1 pack; 3 packs|
OptiTherm™ protein thermal stability kits are used to identify formulations that maximize the thermal stability of protein samples. One kit contains consumable materials to assay solution conditions for up to six different protein samples.
Figure 3. Steps for determining protein thermal stability conditions using OptiTherm™
The OptiResc™ kit is designed to resolubilize aggregated proteins or peptides. The kit contains the same reagents as OptiSol™ and three 96-well mixing plates for visual inspection-based assay. The assay results need to be evaluated by a stereo dissecting microscopy to determine the dissolution of aggregates.
|OptiResc - Protein Aggregate Solubilization Kit||1 pack; 3 packs|
Through systematic solution design and array-based deaggregation technology, OptiResc™ protein aggregate solubilization kits are used to identify conditions under which reversibly aggregated protein samples can be solubilized.
Figure 4. Steps for Solubilizing reversibly aggregated proteins using OptiResc™
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