During the concentration the proteins are aggregated, when it was in the starting stage it showed a lesser amount in OD 260/280, after it was stored in +4'C the result was increased due to the aggregation.
Basically you'll need to test different buffers, varying the pH, the salt concentration and the buffer system itself (some protein are more stable in certain buffers than in others, even at the same pH, e.g HEPES pH 7 might work where TRIS pH 7 doesn't). Also, certain additives can increase protein solubility (e.g. glycerol). One way of testing many conditions quickly is to use dynamic light scattering, this assay can check which of the buffers produce monodispersed protein (a good indicator of protein stability).
Storing your protein at -80oC instead of +4oC might also help. Take a small aliquot and test if it stays in solution after freezing it at -80oC for an hour. Some proteins will precipitate immediately while most proteins will freeze well and can be stored for months and even years at -80oC without loss of activity upon thawing. Again, adding glycerol might help you freeze your protein as it acts as a cryoprotectant. Most of the proteins I have worked with could be stored without the addition of glycerol, but some precipitated after thawing and I found that some of these became stable when I added 5% glycerol to them before freezing them. Lastly, many proteins that are stable after freezing will nonetheless precipitate if they are frozen and thawed several times, so aliquot your protein into small samples and that way you can thaw as much as you need at any one time while leaving the rest frozen.
As Antonio wrote I would check different buffers and additives. You can also test arginine to stabilze your protein. If you want to check the aggregation propensity immediately and you don`t have a DLS instrument, you can easily check the AI at A340*100/(A280-A340). Concentration of your protein may also play a critical role.
test different buffers, pH. You may also want to add glycerol to your buffer.
Can you please confirm whether the proteins that you are trying to concentrate is amyloidogenic? Secondly whether the protein is soluble just in water? If yes, then you can switch between water and the buffer as concentrating medium.
0,1% (v/v) Tween 20 and 5% (w/v) trehalose can help preventing aggregation
This is a difficult situation and the solution is protein-dependent. It basically comes down to testing a range of parameters until you find the right solution conditions that will preserve protein function and minimize aggregation. Some of the key variables are protein concentration, pH, and temperature. As mentioned before, other additives, such as buffer type, salt, and non-denaturing detergents could help. There is no one-size fits all. Systematic testing of different conditions is your best chance. Been there - good luck. If you figure it out, please let us know what worked.
Some proteins might not be compatible with the types of membrane used in the Ultra centrifugation filter, which causes the accumulation of protein clumps. You may try other types of ultra centrifugation filter (eg. Amicon). As mentioned by Antonio Ariza, the addition of 5-30% can prevent the aggregation of the proteins. You may freeze down the protein samples using liquid nitrogen and stored at -80degree. Hope this helps.
Thank you very much to all, i will try and if any doubt i will ask. Actually I am using with 100KDa PVDF membrane as a cassette system. And also i am using with 100mM Phosphate buffer in 100 mM NaCL as a buffer.
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