ATP depletion

[Tiffy]

Hello,
I would like to carry out an ATP depletion with MCF7 cells to investigate a protein translocation into the nucleus. I have found a publication where the authors described a method. But one point I didn`t understand. They used dialyzed serum and I wonder why. Can anybody answer that question? Thanks a lot.
Tiffy
PS It would be very kind if there other advice concerning ATP depletion. What kind of pitfalls etc.

[admin]

Hello Tiffy,
it may have been to alter the pH or buffer a bit of the serum, however can you specify what step they used the serum for?
Also what serum was it FBS (fetal bovine serum)?

Was it for culture of the MCF7 cells?

cheers

[Tiffy]

Hi
thanks for your reply.
The authors (Reilly et al JCB, 2001) used Swiss 3T3 fibroblasts cells but unfortunately they didn`t specify the serum.
They just mention: glucose-free DME containing 0.5% dialyzed serum...
It is a little bit unclear how can I dialyze serum. Against what. PBS? Water? I could imagine that they wanted to abolish a source of compounds which these cells can use to make ATP. Or am I wrong?
Maybe I have to try it without the dialyzed serum. Isn't it practice makes perfect?
Tiffy

[Tiffy]

Hello
thanks for your reply.
The authors (Reilly et al JBC 2001) didn`t specify the serum. They use Swiss 3T3 fibroblasts and they just mention in the methods: ...glucose-free DME containing 0.5% dialyzed serum...
It is not clear how can I dialyze serum. Against what? Water? PBS?
What kind of compound is in the serum what can cause energy in the form of ATP? Am I completely wrong and that is not the reason for dialyze.
Tiffy

[mickey07]

Hi Tiffy

Just a suggestion. Why don't you try and contact the authors of the publication and ask them about their protocol? Hope this suggestion is not too late.
Ciao
Mickey

[Michael C. O'Connor]

I don't know the answer to your question concerning dialyzed serum--- I take a shot at it below anyway--- but regarding the general topic of ATP depletion and translocation into the nucleus, there are not all that many publications on that but I have one for you that discusses ATP depletion, translocation and consequences for apoptosis/necrosis (you asked for pitfalls).

Makoto R. Hara, Nishant Agrawal, Sangwon F. Kim, Matthew B. Cascio, Masahiro Fujimuro, Yuji Ozeki, Masaaki Takahashi, Jaime H. Cheah, Stephanie K. Tankou, Lynda D. Hester, Christopher D. Ferris, S. Diane Hayward, Solomon H. Snyder and Akira Sawa. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nature Cell Biology, 7(7):665–674, 2005

I have not yet made five posts, and so the moderator has anti-spamming policies that don't permit me to give a URL here. Just Google the entire title, and click at the right of the first link that comes up, where it says "all 8 versions". Then click on the 5th one down (counting the indented one), the one from neuroscience_jhu_edu.

I hope that you find the article to be helpful.

Regarding the dialized serum, I found the following from HyClone to be informative:

Dialyzed FBS is used in applications requiring serum depleted of small molecules (less than 10,000 mw) and is filtered through three sequential 100nm (0.1 µm) pore-size rated filters. HyClone has developed a diafiltration process that reduces concentrations of low molecular weight components such as nucleotides and amino acids that are necessary for alternative biochemical survival path ways. The process is reproducible and reduces hypoxanthine and thymidine concentrations below detectable limits. While producing the same results, a major difference between diafiltration and dialysis is that the driving force for diafiltration is hydrostatic pressure, rather than concentration gradients with dialysis. Monitoring glucose concentrations carefully controls the diafiltration process. The glucose concentration, which is representative of the extent of dialysis, is reported for each lot of serum.

Again, this time Google "dialyzed serum definition" and HyClone is the third link down.

[kiki06]

Thanks Michael your information helps everyone welcome to the forums