Collagen generally gets solubilzed in acidic condition. My experiments need collagen to be at neutral pH. But It's really getting difficult as collagen precipitate when the pH was increased.
Can anyone please suggest me the method/condition by which collagen can be in soluble form at neutral pH.
Thank You.
I would suggest to first obtain a dispersion (or colloidal solution) of collagen using citrate buffer (pH ~3.4). After some convenient maceration time, preferably at controlled temperature (cold), the suspension can be neutralized by adding NH4OH aq., in order to obtain a more or less stable dispersion of collagen in ammonium citrate tribasic buffered solution, at nearly neutral pH: (e.g.) approx. 6.5-8.5 for 10 wt% (NH4)3C6H5O7. It may help to add up to about 10 wt% of NaCl to this dispersion, possibly stepwise, to adjust its ionic strength, if not too high. Hopefully, the stability of the collagen dispersion can possibly be maximized at some particularly suitable ionic strength, which could be found by either centrifugation or sedimentation testing, or by viscometry analysis. The concentration of the buffering agent, which may also play a dispersing role, besides contributing for the ionic strength, may also be passible of some optimization. Other soluble neutral monovalent salt(s) could, in principle, substitute for NaCl. Salts with plurivalent cations (e.g. CaCl2) should be avoided, as they may act as flocculants. Of some influence may be the type of collagen, its original raw source, and prior treatments (drying included).
Hello Sneha,
First of all, the crucial question is the purpose of your experiments using collagen. Presumably you have in mind fibrillar collagens which are usually extracted from tendon (fairly pure collagen type I) or from skin (mainly collagen type I and some type III) using weak acetic acid, as you have mentioned. Very little collagen can be extracted from fetal skin also by neutral salt solution. What you call 'collagen powder' probably refers to freeze-dried/ lyophilized material, from such solutions, which dissolves quite easily in water giving slightly acidid solutions. The pH can be adjusted with NaOH solution (pH control!) in the cold/ on ice. Rising the temperature will cause the formation of collagen hydrogels, a common procedure to suspend and grow cells like dermal fibroblasts witrhin this matrix (artificial ECM) and to create a support for surface epithelial cells such as epidermal keratinocytes. If something like that is one of your goals the following papers may be helpful, (i) HJ Stark, M Baur et al., J Invest Derm 112,681-91, 1999 or (ii) R Nischt, C Schmidt et al., J Invest Derm 127, 545-54, 2007 (see also for further references). Please let me know if you have further questions on the ECM issue.
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