[Histonet] rat myelofibrosis questions
Rittman, Barry R
Barry.R.Rittman <@t> uth.tmc.edu
Thu Nov 2 13:04:38 CST 2006
I am a little confused so should put my thoughts down.
EDTA has four possible chelation sites for chelating calcium and other ions.
If you start with EDTA powder and adjust the pH upwards you will first get mono, then di, then tri and finally tetrasodium EDTA.
EDTA powder by itself if not very soluble until the pH is adjusted. Most recipes start with the disodium salt.
I forget the actual pHs but memory is that they are around 4.6, 6.5, 8.5 and 11 respectively (forgive me if incorrect but I am not at my office computer).
Whenever you adjust the pH you alter the EDTA salts that are produced.
So that at pH 7.0 or so you have a micture of disodium and trisodium.
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Gayle Callis
Sent: Thu 11/2/2006 11:50 AM
To: Missy; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] rat myelofibrosis questions
Concentration of EDTA will depend on which EDTA you use.
EDTA, disodium or EDTA without any sodiums attached only goes into solution
at around 10% or 10g/100 ml, pH 7.0 to 7.6 and people often have to use
sodium hydroxide to make it go into solution which should also adjust the
pH up to 7.0.
If you use EDTA tetrasodium you can get the concentration very high at 14%
or more, but you must adjust the pH down. We use glacial acetic acid for
this purpose (bone expert Webb Jee used this EDTA decalcification method
for bone). EDTA tetrasodium dissolved in water or buffer has a pH of
approximately 9 to 11, so the pH should be adjusted down to pH 7.4 or
so. If the pH remains very alkaline, then the alkaline sensitive protein
linkages can be damaged.
You may want to look at the femur too, since you could use the
contralateral side (femur) for your other test. Have you done a
literature search on mouse ilium just to see what is out there. Google
Scholar is a good place to search if PUBMED becomes cumbersome. Sternum is
another bone marrow biopsy site in humans - just a thought here.
We used to do fibrin staining on acid decalcified bone marrow biopsies
without problems rather than use the slower EDTA decalcification.
If you useAt 09:37 AM 11/2/2006, you wrote:
>Sorry to bother again, I just have a quick question. I am looking for
>myelofibrosis in rat, we had intended to look at the femur, however we got
>to talking and brainstorming and were wondering if the ilium would be a
>better place to look. Since the iliac crest is the location of bone marrow
>biopsy in human, and that way we can "double dip" on the animals and use the
>femur for another assay. Do you think it will work or has any one gotten
>positive results using the ilium?
>Also, regardless of wehter we use femur or ilium, which concentration of
>EDTA is optimum for decal?
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