I would suggest to first solubilize the collagen source material by using acidic buffer, rather than just the acid. In case you want to consider the sodium acetate – acetic acid buffer, it should be noticed that its buffering capacity decreases noticeably below pH 3.7.

We could alternatively consider a citric acid – sodium citrate buffer for a lower stable pH, down to 3.0. The pH of the actually used 0.5 M acetic acid would be 2.5, what may be too low; depending on the solubilisation temperature and time, and on the collagen source and purity. Of course, collagen hydrolysis should be avoided.

I would suggest keeping the pH within 3.0–3.8 to solubilize raw collagen. The isoelectric point for fish collagen may be located around 8. In order to precipitate prior solubilized collagen by adding ammonium sulfate, it seems advisable to readjust the pH below but close enough the isoelectric point, possibly by around 7.5, for the considered type of collagen. The collagen should then have net positive charge, so that it could offer cationic sites for the sulfate ion to bind. Salts with plurivalent cations (e.g. CaCl2) can also help to promote precipitation. Adding acetone, ethanol, methanol, tetrahydrofuran (THF), or some other conveniently selected water-miscible solvent, possibly previously cooled, would also favour precipitation. The mother solution should not be too cold. Peptizing agents (e.g. urea) are to be avoided, because they might hinder precipitation.    

Thank you for the answer Carlos Aráujo. I actually tried your suggestions of using two acidic buffers to solubilized the sample, but the precipitation with NaCl/ Amonnium Sulfate didn't work.

At the end the acetic acid 0.5 M and NaCl worked, by making some adjustments to the time and temperature of the solubilization process.

I am working with collagen I from rat tail. When I prepare this collagen in 15 mM NaOH. The solution become trouble so it seems that the collagen precipitated. I would like to understand the chemistry behind this phenomenon.

Please advise

Wafa

Try to use alkaline solutions , like NaOH 0.5 M

Dear Melissa Chaves,

You may use also 0.35M acetic acid and NaCl powder directly to your extracted solution to precipitate the collagen and do a higher centrifugation step. It might be helping you to get a high yield of collagen content. best of Luck!

Dear Wafa Bouhlel,

Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Please refer to this paper for your further understanding. Thanks.

Melissa chaves.. May I please know how did you do salting out with NaCl. What adjustments with time and temperature you performed that salting out occurred