[Histonet] Pas Digestion

[Caroline Miller]

Yes, 37oC is optimal, however an enzyme is often active at 25oC but to a
lower level of efficiency....you just leave it longer.

I don't know if you ever did this in the US, but when I first learned the
dPAS technique we used to spit on the slide :)

Much nicer is to buy some diastase such as this:
http://www.americanmastertech.com/store/main.aspx?p=ItemDetailStyles&item=AHDIA50

and I have attached what we used to do in London (the spitting was only a
good trick to show the trainees)

Maybe it is the freezing? Enzymes don't really like that unless there is
glycerol and /or a high concentration of enzyme. I would just make it up
from powder fresh every time, it is not that hard or time consuming

yours,
mills

On Tue, Nov 3, 2015 at 2:54 PM, Jennifer MacDonald via Histonet <
histonet at lists.utsouthwestern.edu> wrote:

> The optimal temperature for most enzymes in 37 degrees Celsius.
>
>
>
>
> From:   "Lager, Loree via Histonet" <histonet at lists.utsouthwestern.edu>
> To:     "'histonet at lists.utsouthwestern.edu'"
> <histonet at lists.utsouthwestern.edu>
> Date:   11/03/2015 12:38 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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-- 
Caroline Miller (mills)
Director of Histology
3Scan.com
415 2187297