[Histonet] PAS Stain

[koellingr at comcast.net]

Hello 
  
Agree with comments about temperature of 60 degrees killing an enzyme.  If you plot enzyme activity on "y"  axis and temperature on "x" axis it is not a straight line.  Every enzyme has an optimal temperature and can function slowly at non-optimal or optimally at correct temperature.  Diastase/amylase from your body work at 37 degrees, less so or dead at RT or 60 degrees.  Nuking a mixture to 37 degrees might nuke protein.  Nuke buffer to 37, verify temp and add the enzyme. 
  
pH-if you plot enzyme activity on "y" axis and pH on x-axis, it is not a straight line.  There is an optimal pH for each enzyme.  The enzyme might work fine at different pH; just not nearly as well. 
  
I know some still use 0.5% or 1.0% in di water.  I'd advise not to.  Use a buffer at a given pH.  "pHíng" di or distilled water with ordinary lab electrodes is an act of futility.  From a standard lab electrode point of view, there is NO pH to read.  Not enough ions present so meter might say anything.  If anything, it will pick up CO2 from air which becomes carbonic acid and meter will flail wildly in acidic region.  But that is not a stable buffer.  di water might work.  Just that sometimes, it might not.  Why chance it?  di water has unstable (no reliably readable) pH. 
  
Ray Koelling, golfing in Spokane WA and enjoying the low humidity 

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

From: "Joanne Clark via Histonet" <histonet at lists.utsouthwestern.edu> 
To: histonet at lists.utsouthwestern.edu 
Sent: Wednesday, May 4, 2016 1:02:48 PM 
Subject: [Histonet] PAS Stain 

Hi Histonetters, I'm hoping someone can help me troubleshoot our PAS diastase method.  We have been digesting the tissue in 0.5% diastase of malt in a 60 degree oven for 30 minutes, but do not see the glycogen being digested out.  I have tried alpha amylase and beta amylase also without any luck.  Does anyone have any suggestions to get the digestion to work 

Joanne Clark, BAAS, HT(ASCP)CM 
Director of Histology 

P.   (575) 622-5600 
C.   (575) 317-6403 
F.   (575) 622-3720 
TF. (800) 753-7284 

pcnm.com 




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