From the original FM medium paper [PNAS 74(5):2157 (1977)]:
Buffer. The buffer in the medium is the only source of
phosphate. Maximum growth rates occurred at phosphate
concentrations of 1-12 mM and maximum yields were reached
at about 4 mM. Without the phosphate buffer there was no
growth. Since dramatic pH changes occur during growth (Fig.
1), the effect of several "Good" buffers on the growth of the
amoebae was tested in medium M3 (Table 1). The following
buffers, all with pKa in the 6-7 range, were adjusted to pH 6.5
with NaOH or HCL. The numbers in parentheses indicate the
highest concentration (mM) at which growth rate and yield
were unaffected: N-(2-acetamidol)-2-aminoethanesulfonic acid
(Aces) (10), N-(2-acetamidol)iminodioacetic acid (Ada) (2),
[bis-(2-hydroxyethyl)imino]-tris-[(hydroxymethyl)methane]
(Bis-Tris) (5), 1,3-bis[tris(hydroxymethyl)amino]propane
(Bis-Tris-propane) (2), 2-(N-morpholino)ethanesulfonic acid
(Mes) (5), and piperazine-NN'-bis(2-ethanesulfonic acid) Pipes
(5).
Jakob
On Fri, 8 Jan 2010, Robert Insall wrote:
> We have found that the optimum labelling comes by growing cells for a day or so in MES-buffered HL5, then overnight in MES-buffered FM.
>
> A few points:
>
> - MES seems to be toxic in large amounts. Stick to 10mM. I don't know about Bis-tris, never tried it.
> - Any longer than overnight in MES-FM makes cells very sick - not experimentally a good idea.
> - Cells grown in MES-HL5 alone don't label as well - there's a significant amount of phosphate in the peptone - but it would be OK for a start.
>
> I had formedium (see recent other posts) make up a batch of MES-buffered FM. Wasn't too costly.
>
> R
>
>
> On 8 Jan 2010, at 04:22, David Knecht charter wrote:
>
>> Why not use Bis-tris buffered HL5?
>>
>> On Jan 7, 2010, at 3:37 PM, Changhoon Choi wrote:
>>
>>> Hi all,
>>>
>>> I am planning to do metabolic labeling to detect phosphorylation of
>>> some dicty proteins. I found a nice protocol for 32P labeling on the
>>> dictybase web site, but the problem is phosphate-free FM medium. Our
>>> lab is small and doesn't have all chemicals required for FM medium. As
>>> far as I know, all commercial FM media contain potassium phosphate. Is
>>> there anybody that knows any way to get phosphate-free FM medium or
>>> can anybody share some stock solutions with us?
>>
>
> --------------------------------------
> Professor Robert Insall
> CR-UK Beatson Institute for Cancer Research
> Switchback Road, Bearsden
> Glasgow G61 1BD, UK
>
> Tel. (0/44) 141 330 4005
> Web http://www.beatson.gla.ac.uk/robert_insall
> --------------------------------------
>
>
Jakob Franke
424 Tappan Road
Northvale, NJ 07647
Tel. 201-768-3612 home
Tel. 201-638-0582 cell
http://jaep.smugmug.com/
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