For years we have made cell suspensions from sliced organs using the standard EDTA and trypsin method found in many lab protocols. I have always known that many proteases are metalloproteins. I looked up trypsin and found that it requires calcium. It makes sense to get rid of calcium to rob cadherins of their calcium. But, doesn't it make more sense to first treat with trypsin and then treat with EDTA? Also, doesn't it make more sense to treat with EGTA since it is more selective for calcium?
Or, is EDTA useful for reducing the activity of the trypsin.
Hi there,
Trypsin is not a metalloprotease and is actually inhibited by calcium and magnesium. The presence of EDTA is required to chelate divalent ions from the cell culture in order to favor trypsin activity to break up ECM and also to break cell interaction mediated by divalents (cadherin). After trypsinization serum is added to inhibit trypsin (serum contains inhibitors and highlevels of divalent ions).
Hi there,
Trypsin is not a metalloprotease and is actually inhibited by calcium and magnesium. The presence of EDTA is required to chelate divalent ions from the cell culture in order to favor trypsin activity to break up ECM and also to break cell interaction mediated by divalents (cadherin). After trypsinization serum is added to inhibit trypsin (serum contains inhibitors and highlevels of divalent ions).
Hi,
Trypsin does not require metal so EDTA may not block it. Purpose of adding EDTA is inhibit metalloprotease of cells other than trpsin.
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