Is all of this aggravation worth the extra cost that you would dish out
for a commercially prepared DAB? I have used DAB from DAKO, Zymed, KPL,
Biocare, Invitrogen, and Vector, (there are many more out there)and
never had to deal with the issues that you are describing. Most of these
DAB reagents that I mentioned can be prepared in volumes as small as 1
ml, and Invitrogen DAB is ready to use, so you can use as little as
100ul. How much simpler can it get? As my good friend Dr. Laura always
says at the end of her major station breaks: Now go and do the right
thing!
Thank you and have a great weekend.
To anyone that is celebrating a special day this Sunday, have a nice and
enjoyable holiday.
Dusko Trajkovic 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Pixley,
Sarah (pixleysk)
Sent: Friday, April 21, 2006 7:18 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Re: DAB precipitate

Dear Eva:
I would like to hear any answers to this also. We always have a DAB
precipitate. To avoid it, we make the DAB and add the nickel sulfate and
H2O2 immediately before the reaction and do the reaction quickly. (Using
nickel in the solution increases the precipitate significantly.) The
longer you wait, the greater the precipitation. If I am doing a lot of
samples, I set half the DAB aside and change solutions in the middle
(add the nickel sulfate and H2O2). Otherwise, I don't know any other
tricks. 

One other phenomenon I have seen is after freezing DAB. We make up our
DAB solution in phosphate buffer (without H2O2 or nickel) and freeze the
leftover. That works very well as long as you freeze in glass (i.e. new
glass scintillation vials). If you freeze in some plastics, I have seen
a precipitate upon thawing. And the glass has to be very clean--it
cannot be something that was used before for DAB and then bleached and
cleaned. Even if you clean the glass with a good cleaner like Chromerge
or Contrad, there can be enough bleach leached into the glass to
inactivate the DAB.

Good luck and I hope to hear some interesting solutions to your problem!
Sarah Pixley
 

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