Fludarabine

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Fludarabine

Postby Sophs012 on July 13th, 2015, 5:43 am 

Hi all,

I was wondering if anyone has any experience with fludarabine? I want to use it as a STAT1 inhibitor, and it seems to come in phosphate and non phosphate forms and all papers are very vague where they get it from and what form they are using

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Re: Fludarabine

Postby Watson on July 13th, 2015, 10:41 am 

Hi Sophie,
I saw you post this the other day. Sorry you got no reply. I know nothing about this but was sure someone here would. Biowizard may have more to say on the subject.
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Re: Fludarabine

Postby BioWizard on July 13th, 2015, 7:17 pm 

I really thought I had responded to this question when it was first posted. I guess in my rush, I forgot to hit "submit". Thanks for the heads up Watson.

In any case, from what I know, Fludarabine is not an inhibitor of STAT1 in the classical sense. An inhibitor is expected to be something that interacts with and directly inhibits the function of a given protein. Here, Fludarabine downregulates the expression of STAT1. So it indirectly brings down STAT1 activity, but it would be impossible to test hypothesis related specifically to the function of STAT1 using Fludarabine. Fludarabine inhibits DNA synthesis, so it likely affects the expression of a very large number of genes, and the effects you see will likely only be correlated with STAT1 decrease, but not necessarily due to it. If you want to test hypothesis specific to STAT1 function, you'll need to try to knock it down on express a dominant negative, since those are a lot more selective. If it were an enzyme, you could look into finding a catalytic inhibitor. But it's just a transcription factor, as far as I know.
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Re: Fludarabine

Postby Sophs012 on July 16th, 2015, 4:25 am 

Hi BioWizard (and thanks Watson)

From what I have read there have been studies to suggest an inhibition of STAT1 phosphorylation - which is what I want so I will want to see if the phosphorylation is effected, and thus when I incubate neutrophils with IFNs I will see an effect on the downstream pathway.
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Re: Fludarabine

Postby Sophs012 on July 16th, 2015, 4:27 am 

Also, if I want 10uM in 1ml of neutrophils, and I have 5mg of the fludarabine to begin with (MW 365.21) how much would I a) have to resuspend it in initially and b) how much of that to put into the 1ml?
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Re: Fludarabine

Postby BioWizard on July 16th, 2015, 8:36 am 

And how would you know that the effects you observe are due to decreased phosphorylation on Stat1, and not the effect of Fludarabine on thousands of other genes? This is the worst kind of experimental set up to test the function of a transcription factor or it's phosphorylation.

Regarding the volume and concentration of your stock solution, it depends on the tolerance of your final assay to the solvents used to make the stock. In principle, the more concentrated your stock is, the better (since you'll need to end smaller volume to your final assay, which will change final buffer composition less).
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Re: Fludarabine

Postby Sophs012 on July 16th, 2015, 11:32 am 

Because on the western blots shown in said papers show a decrease in phospholation of STAT1 despite IFN stimulation - its a perfectly reasonable experiment for what i want to look at i think
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