The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920 

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, Bradford, etc). If losses are higher than 20-30%, check protein concentration on the flow through. If protein is detected in the flow through, it may be that the unit is damaged or a smaller MWCO should be used. 

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)

Another screening  conditions for agents that may promote protein solubility can be seen in Table1of S.E. Bondos, A. Bicknell Anal. Biochem. 316 (2003) 223–231 (pdf) or in the suggested conditions for small-set buffer/additive screening (Table I) for the differential scanning fluorimetry (DSF) described by Frank Niesen,,
Nature Protocols
(2007) 2: 2212-2021  (pdf)

According to Alexander P. Golovanov et al. (J. AM. CHEM. SOC. 2004, 126, 8933-8939) simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum  achievable concentration of soluble protein. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR  (pdf)


Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,
The Hebrew University of Jerusalem  Tel: 972-2-6586920  

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