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September 26, 2012
Free the Labels
Posted by Derek
When you talking assays, "label-free" is a magic phrase. The more thingies you have to stick onto your molecules or targets to see them, the less confidence you'll have that you're actually looking at the system the way you really wanted to see it: as if you weren't looking at it at all. And while we're not quite quantum mechanics, the observer effect is very real in molecular and cell biology - too many interesting techniques perturb the system in the process of reading out.
And there are no perfect label-free assays, otherwise we'd all be using them. In vitro, NMR can tell you an awful lot, but it can require an awful lot of work if you want to correlate structural information with binding events. And mass spec is getting ridiculously sensitive, and can be used to detect compound binding. But even when that works, it doesn't give you any structure (or much spatial resolution after a certain point, if that's what you're looking for - say, in cells). SPR is a great technique for getting kinetic information right out of the primary assay (instant off-rates!) But it's not quite label-free, because you have to immobilize something to a chip to make it work. Thermal shift is an interesting assay, too - but it uses up a fair amount of protein, and some proteins are more sensitive to it than others. No structural information there, either.
There are a couple of techniques that I don't have much experience with that sound intriguing. Capillary electrophoresis for binding is one - you look at mobility changes with your protein when something is bound to it, as you'd imagine. It's supposed to be pretty sensitive. And BLI (bio-layer interferometry) reminds me a bit of SPR, in that it uses an immobilized protein. I'm not sure what the advantages/disadvantages of that one are, but I see it turn up in the literature.
The ideal assay? If you could do NMR, with the sensitivity to detect very small amounts of a compound, with spatial resolution well below subcellular. You'd get binding, localization, and structure all in one shot. That's probably not even possible, but I'd love to be wrong about that.
Comments (15)
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1. Rick Wobbe on September 26, 2012 7:43 AM writes...
It depends on what kind of assay you want, phenotypic vs. target-based, and within those you have different sub-types (e.g. binding, enzymatic, structural/morphological change), so a single assay that lends itself to apples-to-apples comparisons is impossible.
Having said that, I'm fond of rapid gel filtration chromatography - test compound is in the void (bound) or not (not bound) - for binding assays. Not sexy, rarely employed, but effective, kind of like us old farts.
Permalink to Comment2. MJ on September 26, 2012 8:30 AM writes...
My (limited) experience with SPR is that compared to a lot of other biophysical techniques, it *is* label-free: no isotopic enrichment, fluorescent probe, or spin label required.
Insofar as your ideal assay - my initial thought went to DNP NMR, as while I know they've seen ridiculous signal enhancements in solution NMR experiments with tiny amounts of sample, I don't recall any whole-cell/imaging work off the top of my head.
Permalink to Comment3. Practical Fragments on September 26, 2012 8:42 AM writes...
Practical Fragments did a poll last year on what methods people are using for fragment-based lead discovery. SPR is definitely popular, but it was interesting how many people are using thermal shift.
Permalink to Comment4. Name on September 26, 2012 9:40 AM writes...
Backscattering Interferometry is really the way to go here - no question about it
Permalink to Comment5. HTSguy on September 26, 2012 11:19 AM writes...
An old saying among pharmacologists is "everything sticks to everything else at some concentration". We now know that some of that "sticking" can actually just be coating the protein with many molecules of compound per protein molecule (see Brian Shoichet's work). So one would like binding experiments to yield information about both affinity and the stoichiometry of the interaction. NMR methods that monitor the protein are useful here, ones that look at loss of signal from the free compound - not so much.
Permalink to Comment6. Bryan on September 26, 2012 12:33 PM writes...
My take on SPR and other such techniques involving immobilization is that you are, in fact, labeling your protein. In this case, the label is not a fluorophore or isotope, but the surface to which you covalently or non-covalently immobilize your protein. (That said, I have gotten very good data from SPR so it still is very useful).
Isothermal titration calorimetry is another good label-free binding assay, although like some other assays, it can eat up lots of material.
Permalink to Comment7. HTSguy on September 26, 2012 1:54 PM writes...
In typical SPR, you are immobilizing your protein not to a surface, but to a dextran layer that has a degree of mobility.
Permalink to Comment8. Name on September 26, 2012 2:50 PM writes...
you need VERY little sample for Backscattering Interferometry (nL!!!!)
Permalink to Comment9. Tom on September 26, 2012 2:54 PM writes...
#4 - agree - not quite commercially ready, but should be sometime.
http://www.molsense.com/
Permalink to Comment10. Carol on September 27, 2012 2:46 AM writes...
capillary electrophoresis is a solution based technique that can detect weak affinity (mM) interactions. It is also a separation technique capable of detecting multiple forms of the protein. It is microscale and does not use as much protein as NMR/ITC.
Permalink to Comment11. POCman on September 27, 2012 4:03 AM writes...
The label free version of microscale thermophoresis (from nanotemper) looks to be promising for tryptophan-containing proteins. I've had some very promising results with the labelled versions for proteins that have been very hard to study through other techniques, and the sample requirements are very low.
Permalink to Comment12. Claire on September 28, 2012 3:16 AM writes...
@ 11
Permalink to CommentIt does look good, but at the moment it's really low throughput.
13. Carol on September 28, 2012 5:24 AM writes...
Microscale thermophoresis monitoring the tryptophan of proteins cannot be used for studying protein-protein interactions!
Permalink to Comment14. sepisp on October 3, 2012 4:42 AM writes...
The essential problem with magnetic resonance microscopy is that its resolution is limited by the magnetic field strength and gradient, and consequently, the small field of view. Unless you want to observe the cells of a finger, fitting the patient in a very strong magnet is not practical.
Permalink to Comment15. JB on October 6, 2012 6:50 PM writes...
A combination of those assays in conjunction with mutagenesis data and a crystal structure to support the findings is in my eyes more valuable.
Permalink to CommentCheck out this example:
http://www.ncbi.nlm.nih.gov/pubmed/22982544