Buffer solubility screen to avoid protein aggregation during concentration
Select the proper molecular weight cut off (MWCO) for the protein concentrator. As a general rule the pore size of the concentrator membrane should be two times smaller than the molecular weight of the protein.
Select the concentrator volume size according to your needs. Add some buffer to the concentrator and rinse the membrane. Use the concentrator immediately after washing and avoid drying the membrane.
Concentrate only a small sample before you concentrate the total amount of protein.
Spin at the recommended speed for the particular
concentrator for a few minutes. Check protein concentration (OD280nm,
If the ability of the protein to be concentrated is not known, the concentration should proceed in incremental steps in order to avoid a change in the oligomeric state of the protein to soluble aggregates or insoluble aggregates. Take samples after each step, to check protein concentration and oligomeric state of the protein by analytical gel filtration chromatography (GF). Soluble aggregates can be detected by GF, Light scattering and native gels.
Once you establish the concentration conditions you can concentrate the rest of the protein.
If the protein precipitates at very low concentration, or the percentage loss is great during concentration, perform a solubility screen trying different parameters: different buffer pH; increasing/decreasing NaCl concentration or use additives such as detergents or glycerol, trehalose and others that may help stabilize the protein.
An excellent screening conditions can be seen in the OptiSol Protein Solubility Screening Kit Application
Manual (table IV) from DILYX
Biotechnologies. This screening kit contains a systematically varied
array of buffers (from pH 3 to pH 10) and a series of solubility enhancers
(salts, amino acids, sugars, polyols, reducing reagents)
that enable the determination of conditions under which a particular protein
sample is protected from aggregation or can be de-aggregated. (pdf)
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