Hi, I need to prepare stock solutions for Collagenase I from Sigma (C0130). The provider suggests to prepare solutions in TESCA buffer (50 mM TES, 0.36 mM CaCl2, pH 7.4 at 37 degrees). I tried to dissolve TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, Sigma product T1375) in water but I get a very ugly grey-brownish solution. Sigma says that a "clear colorless solution at 1 M" should be obtained.
Then, when I try to adjust pH with NaOH, a brown precipitate starts to form when I add a considerable volume of NaOH (and the 7.4 pH is still far...). Have you ever prepared TESCA buffer using TES? My TES powder may be quite old...
I've also found recipes for TES buffers with Tris+EDTA+NaCl but I don't understand if it's a different buffer.
Alternatively: have you ever reconstituted the collagenase in HBSS or PBS, instead of TESCA buffer? I read that calcium is needed and maybe I may add calcium to these buffers. Thank you very much!
The buffering agent abbreviated TES is 2-{[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid. If your TES buffer is brown, it should be discarded and replaced because it has degraded.
Tris+EDTA+NaCl might be abbreviated informally as TES for Tris/EDTA/Sodium chloride. This is not what is required to prepare TESCA buffer (50 mM TES, 0.36 mM CaCl2, pH 7.4 at 37 degrees).
I don't know why TES is specified as the buffer for collagenase 1. Perhaps you can use some other buffer. There are multiple options. I would probably try HEPES, along with the required concentration of CaCl2, if TES is not available.
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