[Histonet] Pas Digestion

[koellingr at comcast.net]

As Jennifer mentioned, enzymes have optimal temperatures at which to work.  But also optimal pH.  Is your 0.5% amylase in water or a correct buffer? 
Ray, Seattle, WA 

----- Original Message -----

From: "Loree via Histonet Lager" <histonet at lists.utsouthwestern.edu> 
To: "histonet at lists.utsouthwestern.edu" <histonet at lists.utsouthwestern.edu> 
Sent: Tuesday, November 3, 2015 12:37:04 PM 
Subject: [Histonet] Pas Digestion 

Hello, 

This is my first time using the histonet, as our veteran user retired. 

We've been having an intermittent problem with incomplete glycogen digestion on PASD.  Our digestion solution is 0.5%  α-Amylase, Type VI-B from porcine pancreas (Sigma), which we freeze in small aliquots and thaw for 45 minutes at room temperature before use. We digest for 20 minutes @ room temperature, dropping amylase on slides. 

In troubleshooting the issue, we discovered amylase was several years old, so new amylase ordered.   The new lot (while the same product number) produced even worse results.  After a closer look we saw that the new lot contained ½ of the units/mg solid, 26 to 13, not sure why? 

Anyone willing to share their procedure or suggestions to help us resolve this issue? 

Thank you, 
Loree Lager 
Seattle Children's Hospital 
Histology 




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