Corante

About this Author
DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

Chemistry and Drug Data: Drugbank
Emolecules
ChemSpider
Chempedia Lab
Synthetic Pages
Organic Chemistry Portal
PubChem
Not Voodoo
DailyMed
Druglib
Clinicaltrials.gov

Chemistry and Pharma Blogs:
Org Prep Daily
The Haystack
Kilomentor
A New Merck, Reviewed
Liberal Arts Chemistry
Electron Pusher
All Things Metathesis
C&E News Blogs
Chemiotics II
Chemical Space
Noel O'Blog
In Vivo Blog
Terra Sigilatta
BBSRC/Douglas Kell
ChemBark
Realizations in Biostatistics
Chemjobber
Pharmalot
ChemSpider Blog
Pharmagossip
Med-Chemist
Organic Chem - Education & Industry
Pharma Strategy Blog
No Name No Slogan
Practical Fragments
SimBioSys
The Curious Wavefunction
Natural Product Man
Fragment Literature
Chemistry World Blog
Synthetic Nature
Chemistry Blog
Synthesizing Ideas
Business|Bytes|Genes|Molecules
Eye on FDA
Chemical Forums
Depth-First
Symyx Blog
Sceptical Chymist
Lamentations on Chemistry
Computational Organic Chemistry
Mining Drugs
Henry Rzepa


Science Blogs and News:
Bad Science
The Loom
Uncertain Principles
Fierce Biotech
Blogs for Industry
Omics! Omics!
Young Female Scientist
Notional Slurry
Nobel Intent
SciTech Daily
Science Blog
FuturePundit
Aetiology
Gene Expression (I)
Gene Expression (II)
Sciencebase
Pharyngula
Adventures in Ethics and Science
Transterrestrial Musings
Slashdot Science
Cosmic Variance
Biology News Net


Medical Blogs
DB's Medical Rants
Science-Based Medicine
GruntDoc
Respectful Insolence
Diabetes Mine


Economics and Business
Marginal Revolution
The Volokh Conspiracy
Knowledge Problem


Politics / Current Events
Virginia Postrel
Instapundit
Belmont Club
Mickey Kaus


Belles Lettres
Uncouth Reflections
Arts and Letters Daily
In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

In the Pipeline

« CNN's Cure for Cancer | Main | Those Drag-Over-the-Coals Interviews »

September 26, 2012

Free the Labels

Email This Entry

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) + TrackBacks (0) | Category: Drug Assays


COMMENTS

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 Comment

2. 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 Comment

3. 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 Comment

4. Name on September 26, 2012 9:40 AM writes...

Backscattering Interferometry is really the way to go here - no question about it

Permalink to Comment

5. 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 Comment

6. 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 Comment

7. 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 Comment

8. Name on September 26, 2012 2:50 PM writes...

you need VERY little sample for Backscattering Interferometry (nL!!!!)

Permalink to Comment

9. 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 Comment

10. 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 Comment

11. 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 Comment

12. Claire on September 28, 2012 3:16 AM writes...

@ 11
It does look good, but at the moment it's really low throughput.

Permalink to Comment

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 Comment

14. 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 Comment

15. 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.
Check out this example:
http://www.ncbi.nlm.nih.gov/pubmed/22982544

Permalink to Comment

POST A COMMENT




Remember Me?



EMAIL THIS ENTRY TO A FRIEND

Email this entry to:

Your email address:

Message (optional):




RELATED ENTRIES
How Not to Do It: NMR Magnets
Allergan Escapes Valeant
Vytorin Actually Works
Fatalities at DuPont
The New York TImes on Drug Discovery
How Are Things at Princeton?
Phage-Derived Catalysts
Our Most Snorted-At Papers This Month. . .