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DAB in PBS... How the hell do i dissolve it


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7 replies to this topic

#1 miraclestrain

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Posted 20 August 2009 - 07:09 PM

Hi all,

I am preparing a stain, DAB for my immunohistochemistry study, I am trying to dissolve 0.05mgDAB in 1ml of PBS.
I followed the follwoing steps;
1. took 25mg of DAB dissovled suspended it in 100% ehtanol, warmed the ethanol to 60 deg C.
2. Then crushed it and warmed again for 1 hour,
3. Then mixed it with 1xPBS to required quantity.

DAB is not dissolving in warm ethanol nor later in PBS,
Should i warm it for a longer time or what should I do.

Thanks
MS

#2 HomeBrew

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Posted 20 August 2009 - 07:56 PM

Never used the stuff myself, but in the protocol here, they don't use any ethanol, or heat. They are preparing a 1% (20X) aqueous solution, and have to acidify it to get it to dissolve.

#3 miraclestrain

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Posted 20 August 2009 - 11:03 PM

Hey thanks for that, 80% got dissolved I am waiting for the other part to dissolve. Thanks anyways
MS

#4 pesji

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Posted 21 August 2009 - 03:41 AM

Hi all,

I am preparing a stain, DAB for my immunohistochemistry study, I am trying to dissolve 0.05mgDAB in 1ml of PBS.
I followed the follwoing steps;
1. took 25mg of DAB dissovled suspended it in 100% ehtanol, warmed the ethanol to 60 deg C.
2. Then crushed it and warmed again for 1 hour,
3. Then mixed it with 1xPBS to required quantity.

DAB is not dissolving in warm ethanol nor later in PBS,
Should i warm it for a longer time or what should I do.

Thanks
MS


Taken from SIGMA recommandations !

Buffer pH
will also affect solubility. High pH will deprotonate
DAB to its free base, which is not water-soluble.



#5 HomeBrew

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Posted 21 August 2009 - 04:00 AM

Merged the two threads by miraclestrain on this topic.

#6 miraclestrain

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Posted 26 August 2009 - 07:33 AM

Merged the two threads by miraclestrain on this topic.



I am not quite clear with the pH, High pH would deprotanate the molecule, i believe thats what you have mentioned. So what am I supposed to do?

I have to maintain the pH at 7.4 because I have to stain the sample.

MS

#7 Dr Teeth

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Posted 26 August 2009 - 08:33 AM

Merged the two threads by miraclestrain on this topic.



I am not quite clear with the pH, High pH would deprotanate the molecule, i believe thats what you have mentioned. So what am I supposed to do?

I have to maintain the pH at 7.4 because I have to stain the sample.

MS


Dissolve at a more acidic pH, then return the base to 7.4 with NaOH.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#8 fluffybunny

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Posted 08 October 2009 - 03:10 AM

Dear MS,

Are you still having trouble with dissolving DAB? I use DAB for my IHC study too. However, I bought a dako envision kit which comes with the secondary antibody, the DAB and the buffer as well. Ratio of DAB to buffer is 20 : 1000. Buffer recipe is not enclosed I suppose, but Maybe you can buy the kit? The Dako cat number is K5007. See if Dako just sells the DAB alone.

I'm attaching the EnVision kit's manual in case you'll like to see it.

Good luck!

Attached Files






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