Hi Rikeshwer,

you could also do ITC experiments.

Regards, Stefanie

X-ray crystallography

Today, around half of the detection techniques used are based on luminescence detection. That enables higher throughput measurements, without problems from radioactivity. Probably the most widely used method is FRET, which needs two label molecules. The HTRF technology is improved FRET method, which uses time-resolved detection. Also single-label technique, like the QRET technology can be used as an alternative for radioactive labels. The QRET method uses soluble quencher molecule which acts as a energy transfer partner, but only one labeling reaction is needed. Newest methods are label-free, but the technology is still experimental.

As I previously indicated in a previous post, this a list of experimental techniques that can be used to assess protein-ligand interactions. I am sure many more techniques can be added to this list. Isothermal titration calorimetry and surface plasmon resonance can be considered the gold standard for purified proteins.

Dialysis

Ultrafiltration

Ultracentrifugation (sedimentation equilibrium and velocity)

Chromatography (size exclusion, affinity)

Membrane-filter binding

Capillary electrophoresis

Gel-shift electrophoresis

UV/Visible spectroscopy

Fluorescence spectroscopy (correlation, intensity, polarization, anisotropy, FRET)

Circular dichroism

Dynamic light scattering

Nuclear magnetic resonance (HSQC, STD)

IR spectroscopy

Raman spectroscopy

Electrochemistry

Isothermal titration calorimetry

Differential scanning calorimetry

Surface plasmon resonance

Hydroxyl radical foot-printing

Protease-digestion protection

Mass spectrometry

Atomic force microscopy

X-ray diffraction

X-ray absorption fine structure

Electron microscopy

Biological activity (e.g. enzymatic reaction)

Chemical cross-linking

Two-hybrid systems

Co-precipitation

Western analysis

Proximity ligation assay

Fluorescence microscopy (correlation, FRET)

Flow citometry (FRET)

Confocal microscopy

and surely more...

There are quantitative and qualitative techniques (that is, some techniques give you a YES/NO answer, while other even provide you a number for the interaction affinity, enthalpy,...); some techniques require further modifications of any of the binding partners, whereas other techniques do not; some techniques require milligrams of protein, whereas other techniques may do well with micrograms; some techniques are cheaper/faster/easier than others; some techniques require specialized and expensive equipment, whereas other techniques employ standard equipment...

It is up to you and your circumstances...

fluorescent labelling has the incommode of inserting a fluorescent dye in the receptor which can change its binding properties but has the advantage that it can be couple with other fluorescent observation. So better or not depend of what you want to study.

In the following article, you can see a use of fluorescent binding simultaneously with an intracellular calcium response:

Subcellular Compartmentalization of Activation and Desensitization of Responses Mediated by NK2 Neurokinin Receptors*

Jean-Yves Vollmer‡§, Philippe Alix‡¶, Andre´ Cholleti, Kenneth Takeda**, and Jean-Luc Galzi‡§§

Hello Rikeshwer.

You have a lot of posibilities/alternatives for that. It depends mainly from the system you want to study. Is it more in a cellular environment then why not thinking about kinexa from Sapidyne (https://www.sapidyne.com/introduction-to-reversible-binding.html) or the ligand-tracer aproach from kbc (http://www.kbc.umu.se/platforms/bicu/ligand-tracer-instrument.html). In case of a more soluble or in solution system you have a lot of techniques in fluorescence spectroscopy (FL anisotropy,FL lifetime, FRET and all those derived techniques HTRF etc.) but also thermal shift assays using a fluorophore (DSF) and all the SPR technologies ("biacore like"). Kinexa works as well in these systems. I need a bit more information about your system in order to give you a precise answer. Please feel free to contact me by e-mail : [email protected]. Best, Alain.

In cases where a radioabelled ligand is unavailable or unfeasible but you need to/wish directly study receptor-ligand interaction, the possibility exists to use fluorescent ligands. Until now this approach has been used for only few receptors, alpha-1 adrenoceptors are a good example. For pioneering work in this regard look at the work of the group lead by McGrath (e.g. J Pharmacol Exp Ther 286: 984-990, 1998 or 294: 434-443, 2000).

Dear Martin Michel,

I have fluorescent ligands and i want to do this study with angiotensin receptors. Please let me know that this method is feasible to angiotensin receptors or not?

Despite knowing the angiotensin literatuer quite well (Pharmacol Rev 65: 809-848, 2013), I am not aware of studies applying this approach to angiotensin receptors. However, I see no reason why this should not be feasible. Hopefully the McGrath papers will give you guidance how to set this up. Actually, if you can make it work, that may even generate a nice little methodological stand-alone paper.

We normally use a "mixture" between fluorescent-labelled ligand and intracellular responses, so you know that the ligand not only binds but also triggers the receptor's downstream pathways, such as cAMP, Ca2+, etc. We have not done it for ligand/receptor interactions, rather for receptor-receptor ones, but may be DuoLink would be a good alternative.

BW

A

You can also add the following techniques in order to investigate protein-ligand interactions:

- flash photolysis (if the protein has got a wavelength absorption suitable to be monitored while adding the ligand);

- stopped-flow spectroscopy: you mix the protein and ligand solutions at suitable concentrationsand you follow the spectral changes;

Best wishes for your research work

Ivan Husu

We have good experience with FITC-labelled ligand binding. The assays are at least as sensitive as what can be measured using radiolabelled ligands. Sometimes even more sensitive.....

Dear Rikeshwer Dewangan,

if you have fluorescent ligands available, it would be very straight-forward to use MicroScale Thermophoresis (MST)!

Following this link you will find a very nice review including some MST data on neurotensin:

http://www.nanotemper-technologies.com/technology/publications/article/methods/

Please contact me via [email protected] if you have any questions or if I can provide you any additional information!

@ Adrian Velazquez-Campoy:

this is a quite good summary, but on your list, MircroScale Thermophoresis is missing! :-)

I have to admit, I'm a little bit biased, but I would assign this technology a "Platin"!!! MST has a really huge application range (basically it can detect binding between any kind of (bio-)molecules), without immobilization of molecules, with low sample consumption and it's straight-forward in handling!

Explore it and schedule a demo in your lab with your samples!

http://www.nanotemper-technologies.com/contact-sales/demonstrations/article/mst-information/

@ Heide,

Many techniques are missing from the list I wrote. I am sure MST is perfectly suited for protein-ligand binding studies. I will take a look... Thanks for bringing this up.

I have worked on angiotensin II for 25 plus years. If your goal is to do ligand receptor binding assays, the best way is still to use radiolabeled ligands. However, if you don't like to use radioligands, then fluorescein-labeled compound is the way to go. Check the relevant literature on the subject.

There are many "options", but none of them is as good, sensitive, and precise as radioligand binding. ITC requires tons of protein (low sensitivity), whereas SPR requires a lot of material and one of the partners mus be covalently attached, which introduces lots of artifacts. You should consider other methods only if there is no way to do radioligand binding.

I didn't see Kinetic Exclusion Assay on the list.. The KinExA platform offers a way to study molecular interactions without modifying either binding partner.The assay can be conducted in solution without restriction of buffer/media selection. This technique offers physiologically relevant binding constants.