Each data point is a fraction from one of the CsCl gradients he fractionated the two GFAJ-1 DNA samples on (one for the -As/-P DNA and one for the +As/-P DNA). The -P condition is actually 3 µM added phosphate - this gives growth to approximately the same density as Wolfe-Simon et al's '-P' condition.
The lines with the solid symbols show the amount of DNA in each fraction - these each show a nice DNA peak at around the 800 µl position in the gradient.
The lines with open symbols show the amount of arsenate in each of these fractions - these lines are hard to see because they're sitting right on top of the X-axis (yes, that means that the amounts of arsenate detected are ~ zero 'ion counts'). The real values aren't necessarily zero, but they're below the detection limit for this experiment.
The dashed line shows the amount of arsenate that should have been detected if 4% of the phosphate in the DNA had been replaced by arsenate, as predicted by Wolfe-Simon et al's gel analysis (data in their Table S2).
The second graph shows his standard curve for arsenate detection.
Completely off topic so I apologise. But I thought you might know. Have you had any trouble from journals on submitting a paper if included data has already been published on your blog. Or do they see it as something akin to a conference presentation. Would like to get started but it is a concern. A blog that is. Well, more journal articles would also be of benefit.
ReplyDeleteThank you. Sorry again!
Ewen
Hi Ewen,
DeleteI've been describing my research openly on this blog for about 6 years and haven't had any problems getting the papers published.
Fantastic Rosie!
ReplyDeleteNaturally, I was very skeptical of the initial claims, but it is nice to see this evidence clearly refutes the claims in the paper and press conference.
It looks like the 4% line is theoretical and doesn't represent an actual positive control (given there are no points)? Was a blank AsO4(3-) run as a positive control? Though the intent is clear, might want to relabel that y-axis with either AsO4 or AsO4(3-), as it is a single negative charge doesn't make sense...
Duh, it's the second graph. Apologies.
ReplyDeletehey rosie i might be confused but how is a standard of inorganic arsenate supposed to be help you quantify arsenate that might be tied up in esters in the DNA (which would obviously have a different mass). Does your mass spec approach fragment the DNA as it flies so that you would see any incorporated As atoms appear at the same mass as in free arsenate? If not (that is, arsenate would be expected to be tied up in the DNA in the unlikely scenario that As-DNA is real), then its hard for me to understand how your 4% incorporation threshold, based on free arsenate, would make sense. Just looking for a little bit more detail on the type of mass spec you performed and how those experiments were performed (perhaps your colleagues [aka 'the graduate student' ;) ] could help fill us in if you aren't sure on all the details).
ReplyDeleteGood luck getting this ready for publication! We are all so excited for you :) :)
This is great! The old-timers in my lab (HOS, CHIII) appreciated when I remarked that a CsCl gradient would solve this problem. I know you're not interested in doing more experiments, but I think it would be really strong result if you ran sequencing on the samples, did random priming, measured read lengths, and put an upper limit on the Arsenic content, assuming a nicked DNA strand would halt a read.
ReplyDelete