Hi Shashikiran, your protein could be precipitating because it has a pI close to the pH of the tris buffer.

It could also be precipitating because you are concentrating it too much without using a stabilizing agent.

Many proteins will precipitate at higher concentrations, hence as the protein gets concentrated in the concentrator (obviously), it will precipitate.

Also, does your protein need NaCl  for stability? NaCl is being removed,  while concentrating  (these concentrators are also used for desalting of samples),  hence if NaCl is important for the stability of your protein, desalting may cause it to precipitate.

Try adding stabilizing agents like Glycerol at least 1 to 2 % in ur FPLC Buffer.. But if u have experiments like CD its not a good idea..

Possibly there are a lack of a protean factor with low molecular weight (or enough higher for being discarded during elution profile sampling) or any ionic component loose (gel filtration provokes desalinization) who prevented interaction between interest protein units or subunits, thus aggregation (for example, when hydrophobic patches are more exposed, repulsion ionic interactions disappears, etc.), denaturalization due to elution condition (if elution buffer if different from sample preparation buffer), dimerization, or polymerization due to redox environment changes through S-S bond formations is happening. Protein concentration may favor those processes. Take a look at literature if there is possible to find natural stabilizer or mechanism of functioning that may help you selecting eluting buffer conditions and additional components for preventing precipitation.

For example you may conduct a fast protein precipitation assay with (NH₄)₂SO₄ and if a significant amount of protein precipitate is observed, compared with ionic strength values after gel filtration when low salt concentration is added, then ionic strength must be readjusted, and later must be specifically adjusted with specific salts content presented in buffers. You may test imidazole for discarding it.

You may express salt concentration as a % and measure protein concentration in supernatant spectrophotometrically measured at 205 nm if DNA and proteins are mixed where C(prot) mg/ml= 31*205 nm, or C(prot) = A280 nm - A260 nm or at 220 nm, because no great protein specificity or accuracy is needed for this preliminary information when all you need is a precipitation profile.

More questions or comments? Do not hesitate to contact me!!!