One of the three newly published protocols is for the microfabrication of anisotropic nanofluidic sieving structures. This protocol opens the possibility of a more direct analysis of biological molecules by continuous flow separation as it uses nanofilters of precise, macromolecule-size dimensions. And of course everything done on this tiny scale is pretty cool. The two types of ANA described are referred to as ‘planar’ and ‘vertical’. They are schematically illustrated in Figure 1 and there are some amazing photographs in Figure 2. You can also get a flavour for the protocol, from the procedure for the fabrication of the vertical ANA device (Box 1).

This is one of the first protocols that I have been responsible for since my return, and so I can tell you that its publication in Nature Protocols was dependant on the addition of this sentence to the abstract (and the expansion of the ideas to the introduction):

“This protocol is most useful for bioengineers who are interested in developing automated multistep chip-based bioanalysis systems and assumes previous cleanroom microfabrication knowledge.”

In other words, you can’t just wake up one morning and decide that you would like to make some sieves-on-a-chip, and expect to be able to set it up: you need to have some relevant experience and you have to have a very, very clean room.

The Dekker household recently acquired a new vacuum cleaner: the type that can also be used to clean your carpets and suck up large spills/floods of the type that have started to loom large on the event-horizon. The first time I used it… well it is astonishing how much dirt is hiding in the carpets, isn’t it? But I thought: perhaps our old vacuum cleaner was not really doing its function and next time there will be less. The surprising thing was that even if I vacuum every day, it still picks up an extraordinary amount of hair (inter alia). We must shed it all the time.

Now, I know that there is a lot more to a cleanroom than keeping stray hairs out, but it is amazing to think that the structures on these chips are so much smaller (0.075 – 1.2 microns) than the diameter of the hairs on my head (diameter of human hair is apparently 40 to 120 microns).

When “Work Blog” started, the idea was that I would write to “my mother” about what I was doing at work. Well, I have been wracking my brains, and the best that I can come up with at the moment is something that I am supposed to do every second Monday:

Provide a list of newly published Nature Protocols that can be added to chemistry@nature.

These protocols get added to the chemistry e-alert that is sent every second Friday, and are archived in the research collection under a list of handy keywords. When I was at my most prolific (editorially speaking) and when Baldo Lucchese was still working with us (in 2006 and 2007), Nature Protocols contributed loads and loads of manuscripts in the synthetic category, but now most of ours that are relevant probably find their way into biochemistry.

I notice, looking at the homepage for chemistry@nature, that there is also a Chemistry Podcast! Goodness me! Things have really moved on in the last year – I must try to listen to that at home!

There is an awful lot of information that can be obtained from an nmr experiment and, while the physics behind it makes my eyes go a bit squiffy, I think that it is all very cool.

The latest published protocol on protein nmr is: Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy and is useful for investigating the structures of the excited states of proteins. These conformations are usually “invisible” because they are short-lived and the proportion of molecules in them at any one time is very small.

The Procedure deals pretty much exclusively with the methods for preparing the labeled protein samples required for the NMR experiments (summarised here.

The NMR part of this protocol includes the Carr – Purcell- Meiboom – Gill (CPMG) relaxation dispersion NMR experiment. Now, as we all know, being able to type a name like that in a blog post does not mean that I have the slightest clue what it is. And, in this case, I did not.

So, Google to the rescue. My searches led me to the following web resource:

Advanced NMR web course – page maintained by Françoise Sauriol, and the page where the explanation for CPMG rests is that that deals with Spin-Spin Relaxation time (T~2~). I started to relax somewhat: those are all words that I can understand, and it jogs a memory regarding MRI scans… I did a google search to remind me, and the Wikipedia entry seemed to have the best description, though it could probably do with some more expert attention.

Another search led me to: The Basics of NMR, an electonic book by Joseph P. Hornak and I quickly read through the first three chapters. I think that the important thing to notice is that the decay of transverse magnetization is affected by molecular interactions experienced by the nucleus in question.

Okay, okay. Back to the question at hand (and the NMR web course page). Scroll down to CPMG. Hmm. Scroll up to Hahn Spin-Echo. Back down to CPMG. Gosh it seems complicated. Again, I think that the important thing is that the CPMG experiment allows us to get a “number” (the size of the echo) that is affected only by the spin-spin relaxation process of each nucleus (having the chemical shift associated with the radiofrequency of each pulse).

So we are looking at changes in the molecular interactions experienced by nuclei in the protein molecule.

The protocol itself does not have any figures (lots of tables though), but there was a publication by Prof Kay’s group in Nature a few years ago, and these figures might be illuminating. Follow the links!

There has been a moment of great excitement at my desk: There is a Network Protocol, based on a Nature paper, that has a query from a reader, and a reply with useful information.

Counting Marine Microbes with Guava Easy-Cyte 96 Well Plate Reading Flow Cytometer

" Wonderful and straight forward however is the SYBR green the one supplied by invitrogen? In which case is the working concentration 5x?

Thank you

Posted by: Ssemakalu Cornelius | June 8, 2009 01:15 PM

Yes, we use Sybr Green from Invitrogen. We use a 1:20 or 1:30 dilution of the stock and stain cells at a 1:100 dilution so it is a final dilution of 1:2000 or 3000. It is possible that the 1:3000 dilution gives less background noise. We stain for at least one hour before counting.

Posted by: Kevin Vergin | September 25, 2009 09:38 PM "

While this is not the first useful discussion on our site, these exchanges are sufficiently rare for it to be a cause for celebration!

This week, a new page has been added to the Nature Protocols website!

Stem Cell Series

Peer-reviewed and user-uploaded protocols from our site are sorted under various headings like: “Isolation and Preparation”, “Differentiation of stem cells to specific lineages”, and “Tissue regeneration”. Induction of pluripotent stem cells from fibroblast cultures is the free featured protocol on this page.

Our new featured protocol (free for this week) is:

Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates

There is also a vacancy for Chief Editor!

You can find the advert here.

I think that Nature Protocols is an especially good journal to work for if you have made a move away from bench-research, but enjoyed and ever-so-slightly miss mucking about in the lab. Also: It is an exciting opportunity if you are interested in playing a role in shaping the on-line article of the future (that has a print version to help make it so that the intellectual steps are not lost).

As almost all of you know, it was the Science Online conference in London yesterday. For some reason, I thought it was in September until I saw Richard Grant sitting in the reception area at Nature. This is the type of thing that never happened to me before Baby Hap came along.

Anyway. I obviously hadn’t registered, but was aware that I could go along in Second Life. I was full of confidence about this, as I am one of those rare people for whom the whole Second Life thing has gone quite swimmingly. You can imagine my horror, when I spent the first few hours on Friday night trying to work out why our internet crashed every time I entered Second Life. After much googling and scrolling through JIRA pages, I came across this one suggesting that I disable Voice/Chat.

This worked!

I then fiddled about with my appearance. Going with an avatar that is something close to reality means that when you have put on weight and aged a bit you might feel a moral obligation to transfer these changes to your on-line persona.

Ms Pizzicato attended the conference on Saturday…

… while I listened to the talks and encouraged Hap to play with her new laptop.

It was good to be part of and follow the event in real-time, but if you have the discipline to put aside the “amount of time” in your own time, you would probably get more out of reading and contributing on the various blogs and friendfeed/twitter threads than I did on Second Life.

Here are some links:
the mind wobbles has excellent blog summaries of the talks

There is some discussion on friendfeed.

Apparently there is more on twitter, but I have trouble following conversations on twitter generally…

(all of the titles in our content)

(this is for the protocol that is currently featured: Spheroid-based human endothelial cell microvessel formation in vivo)

I had better get back to it now.

In general, I really enjoy my job: it is stimulating, I get to chat to interesting people, and the stress level is pretty minimal (let’s face it, no lives are in the balance). But there are times when I wonder what the point is.

…In a moment of anger I wrote in some detail what had upset me. A few kind words of caution (including Richard’s comment below) and the passage of a few hours have made me realise that it was wrong of me to communicate this in such a public place…

But, perhaps it as well to know that the life of a lowly Nature Protocols editor occassionally has a stormy teacup. Just in case any of you thought that it was all sweetness and light.

This afternoon, I will channel this vigour into a really promising looking manuscript, and hopefully have a bit of time to do some commissioning-type research.

I realise that it is also bad form to write a post and then delete the important part, and for this I apologise.

My understanding of principal component analysis is Wobbly. It’s a good understanding but it Wobbles…

This tutorial seemed to help: PCA Tutorial mostly because it did not lean too heavily on the maths, the language was clear and they gave a worked example.

I have a suspicion, though, that I am going to have to look PCA up every time I have a protocol that touches on it. Part of me feels that this is like needing to look up the definition of standard deviation every time it comes up, but having said that… I would probably only be able to give you the correct equation straight away on a Very Good Day.

I suppose that there are a lot of brilliant people for whom this is not true: but for me, at least, maintaining my understanding of “concepts” requires quite a lot of time. Otherwise, it wobbles. It’s good, but it wobbles.