by Jessica Helweg | Sep 16, 2019
Yes and No. The most effective inhibitor of protease activity is to use serum-containing media. Adult serum is more effective than fetal serum. The protease activity is likely inhibited by alpha-2-macroglobulin. Buffers or media containing human serum albumin (HSA)...
by Jessica Helweg | Sep 16, 2019
The initial fermentation procedures to generate collagenase or other bacterial enzymes required the use of mammalian meat peptones that contained collagen peptides. As collagenase fermentation methods were refined, gelatin peptones were used in place of meat peptones....
by Jessica Helweg | Sep 16, 2019
Yes. The supplemental protease used for cell isolation is determined by the precedent set by the enzyme suppliers. However, it is likely that other proteases will also work but it needs to be determined by experimentation. Three different proteases have been used to...
by Jessica Helweg | Sep 16, 2019
Yes. Lot qualification is required because of the difference in enzymatic/biochemical compositions between different lots of poorly defined, crude or enriched collagenase products. Replacing poorly defined collagenase with defined collagenase usually requires a single...
by Jessica Helweg | Sep 16, 2019
C. histolyticum collagenase and many other bacterial neutral proteases are metallo-proteases. They require excess calcium to maintain their enzyme activity. One mM calcium ion is sufficient to protect this activity. Cation chelating agents such as EDTA or EGTA should...
by Jessica Helweg | Sep 16, 2019
No and yes. For poorly defined collagenase products, many isolators use these values as a guideline in selecting a specific lot of collagenase. These analyses are an unreliable predictor of successful cell isolation because the collagenase is often degraded by...
by Jessica Helweg | Sep 16, 2019
Each lot of poorly defined crude or enriched collagenase is unique because it represents the enzymatic activities present in the C. histolyticum culture supernatant. The enzyme activities listed on the Certificate of Analysis can be used as a guide but they are not...
by Jessica Helweg | Sep 16, 2019
Initially, crude collagenase manufactured and sold by Worthington Biochemicals in the early 1960’s and later by Sigma-Aldrich. These products are minimally processed bacterial culture supernatants. Later, Sigma manufactured Collagenase Type XI, an enriched collagenase...
by Jessica Helweg | Sep 16, 2019
Nearly all collagenases used for cell isolation are derived from culture supernatants recovered after anaerobic culture of Clostridium histolyticum. A rich broth containing digested animal meat is frequently used to ensure growth of C. histolyticum. C. histolyticum is...
