higher molar Sodium chloride (300mM) can influence the protein aggregation during the chromatographic elution?
Hi there,
The effect of salt concentration on protein solubility is dependent on the nature of the protein (what is the nature of the side chains which are solvent exposed?). The overall effect is quite the same : at lower salt concentration, salting in effect which increases protein solubility and then salting out effect at higher concentration (promoting protein precipitation) but the range of salt concentration to consider to observe these effects do vary a lot from one case to an other... As you mention 300mM NaCl, I would say this is high salt concentration for some and low salt concentration for others...
The ionic strength of the medium and therefore the salt concentration impact the aggregating properties of proteins. Using buffers with 300 mM NaCl is very common in many protein purification protocols and does not adversely affect most proteins but the effect should be analyzed on each protein and it is difficult to predict in beforehand.
An easy way to detect if aggregation is to perform a scan of absorbance between 220 and 450 nm. If the signal is not equal to zero between 320 and 450 nm and the signal increases linearly when decreasing wavelength in this range then, it can be some aggregation.
Hi there,
The effect of salt concentration on protein solubility is dependent on the nature of the protein (what is the nature of the side chains which are solvent exposed?). The overall effect is quite the same : at lower salt concentration, salting in effect which increases protein solubility and then salting out effect at higher concentration (promoting protein precipitation) but the range of salt concentration to consider to observe these effects do vary a lot from one case to an other... As you mention 300mM NaCl, I would say this is high salt concentration for some and low salt concentration for others...
Hi Selavraj,
The effect of ionic strength is highly dependent of protein you work, but a point that would pay attention is the presence of surface charges (patches) and/or the global content of aspartic and glutamic residues. If there is a high density of acidic residues, the increases of salt will stabilize your protein...
thanks to Diago, Dominique Liger and Juan Martinez, if the higher salted protein can elicint higher immune response?
about your first question (salt and aggregation), the degree of aggregation can change, depending on the protein. Here an example with the protease inhibitor BPTI:
Lafont, S., Veesler, S., Astier, J.P., Boistelle, R. (1994) Solubility and prenucleation of aprotinin (BPTI) molecules in sodium chloride solutions. J. Cryst Growth 143, 249-255.
First consider the nature of the aggregation process. Is it driven by an electrostatic or hydrophobic interaction. If electrostatic then ionic strength will help to prevent that. However, if it is due to hydrophobic interaction then salt would promote aggregation of molecules. In this case glycerol might be better.
Dear Chrisostomos Proddromou, I am not clear about the glycerol can act as protein aggregation prevention,let me know the detail, waiting constructive ,.
The mechanism by which glycerol works is not fully resolved. We know that glycerol can stabilise protein fold and prevent hydrophobic surfaces from interacting. I came across this reference that might shed more light on the matter:
Biochemistry. 2009 Nov 24;48(46):11084-96. doi: 10.1021/bi900649t.
Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.
Vagenende V1, Yap MG, Trout BL.
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