Reduce protein concentration. The protein might be precipitating for two reasons: the pH is set to the pI value and the presence of chitosan. You need to lower the pH of the chitosan solution to 4 if possible and then add the protein solution. See what happens. You also need to check that your protein is soluble at pH 2, otherwise you will need to increase the pH of the tpp solution.

Reduce protein concentration. The protein might be precipitating for two reasons: the pH is set to the pI value and the presence of chitosan. You need to lower the pH of the chitosan solution to 4 if possible and then add the protein solution. See what happens. You also need to check that your protein is soluble at pH 2, otherwise you will need to increase the pH of the tpp solution.

 Hi Deepjyoti, there are lots of control experiments you need to do before you can draw any conclusions:

1) "c) The protein solution (in pure water) is now added dropwise to my chitosan solution. (Observation: The solution turns milky white, which I guess is due to denaturation of the protein)"

If the protein is soluble at pH 5 and the chitosan is soluble at pH 5 (which it shouldn't be), then there should be no change in the solution when they are added together, unless the protein has a higher pH, which will cause the chitosan to aggregate and turn milky.

My guess is that the protein solution you are adding has a higher pH than the chitosan solution and making the chitosan aggregate without trapping any of your protein. Do a control of just the buffer and see if he chitosan turns milky.

Another consideration is the quality of your chitosan. Chitosan has many free -HN2 groups and should be insoluble at pH~5. Are you working with chitin instead of chitosan?

Dear Dr Omar Gonzalez-Ortega and Dr Steingrimur Stefansson,

Thank you for your time. I will take your valuable comments into consideration. As Dr Stefansson has asked, I am using low molecular weight chitosan in making the NPs.