What is the initial buffer where the protein is dissolved? Anyway, there is no straight answer as it depends on the particular protein. Move away from the isolectric point. 10% glycerol makes me assume that your protein was initially solubilized from inclusion bodies. Is this so?

For some proteins, there is a limit to the concentration that can be achieved under a given set of conditions before aggregation and/or precipitation occurs.

Changing the conditions may allow a higher concentration to be achieved. Some simple things to try are changing the pH and the salt concentration. In some cases, it may be preferable to keep the protein at room temperature instead of the usual preference for keeping it cold.

It is also worth considering whether it would be sufficient to keep the protein concentration lower than usual to minimize self-association.

If the protein aggregates because of hydrophobic interactions, including a detergent such as CHAPS or dodecylmaltoside may be helpful.

Dear Hossein

to provide a reilable answer we need to know more details about the properties of your protein, eg

- which is the isoelettric point,

- Do it contain cisteines

- Do it contain hydrophobic regions?

and about the purification process that you perform before dialisys

- Which was the protein extraction and purification buffer?

best regards

Manuele

If you don't have the resources for a detailed screen I would try L-Arg and L-Glu at 50 mM first. It's tolerated in most assays, solubilizes many proteins, and not too difficult to remove if necessary.

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Manuele Martinelli

My protein theoretical pI: 9.28 contains six cysteines (3.3%).

The protein extraction method is nickel column and dialysis buffer is 20 mM Tris buffer pH 8.0 that contains 150 mM NaCl, 10% glycerol.

Dear Hossein Ansariniya

2 more questions?

1)Was the protein expressed in E.coli=? Cytoplasmic expression?

2) Which lysis buffer did you used? The buffer contain reducing agent as DTT?

Because i think that one possibility is that your protein aggregate via intermolecular S-S bonds formation due to the fact that the some of the 6 cysteines do not form correctly the S-S bonds.

In this case you have to possibilities:

1) Optimize your expression protocol (eg using an E.coli strain as Origami the promote S-S bond formation of add an N-terminal periplasmic leader peptide to produce the protein in the periplasm)

2) Add DTT or TCEP in the dialisys buffer. But in this case is possible that you do are able to maintain the protein soluble but in a not folded and active form becasue the reducing agent will destroid the S-S bonds, so i do not like a lot this possiblity.

Ciao

good luck

Manuele

Manuele Martinelli

Thanks for your constructive comments, Dr. Manuele Martinelli

Yes, it is expressed in the cytoplasm of E. coli.

Lysis buffer contains Tris-Hcl(20mM & pH=8), Nacl(150mM), Triton X100 (0.1%), glycerol(10%), imidazole, lysozyme(200μg/ml) and PMSF.

Could try 0.05% Tween 20 and/or 0.1% PEG-8000. For some proteins Tween works better than Triton-X, and PEG helps block hydrophobic regions.