HTRF simplifies protein-protein interaction biology

The human interactome has been estimated to cover approximately 400,000 protein-protein interactions (PPI). Understanding and modulating these biomolecular complexes is one of the foremost challenges in the discovery of innovative drugs for pathologies related to oncology, inflammation, CNS, metabolism, and infectious diseases, among others.

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Let’s probe unlimited PPI applications.

Possibilities are unlimited, including Receptor/ligand, Nuclear receptor/ligand, Protein/DNA, Protein/RNA, Protein/peptide, Protein/carbohydrates, and many more.
Let’s see how researchers took advantage of HTRF to build various PPI assays.

Virus blockade assay

Adapted from Newton et al. / Journal of Biomolecular Screening 18(3) (2012) 237–246

PPI Receptor-Ligand binding assay

Adapted from C J. Rossant / Journal of Biomolecular Screening 2015, Vol. 20(4) 508–518

mRNA Protein binding assay

Adapted from M.D.Disney et al. / ACS Chem Biol. 2012 October 19; 7(10): 1711–1718

Antibody epitope mapping

Adapted from C J. Rossant / Journal of Biomolecular Screening 2015, Vol. 20(4) 508–518

Protease assay

Ingo H. Engels et al. / Analytical Biochemistry 390 (2009) 85–87

A wide range of biomolecular interactions can be properly assessed.

The advances in identifying short primary sequences mostly involved in the PPI interfaces, also called hot-spots, have greatly facilitated the setup of biochemical assays to identify PPI modulators. However in some projects the binding epitopes involved in PPI interfaces are not fully understood, which requires the use of full size proteins to reproduce the interaction and screen for inhibitors or stabilizers.

HTRF is the technology of choice for PPIs, whatever the molecular weight of your protein partners, as illustrated below :

Large size complex
B-Catenin/TCF4
This study was performed to show the ability of HTRF PPI reagents to address large biomolecular interactions. Recombinant human GST-β-Catenin (115 KDa) and 6HIS-TCF4 (55 KDa) interaction was detected using Anti GST-Eu Cryptate (150 KDa) and Anti 6HIS-d2 (150 KDa) antibodies.

Read this note to see how successful HTRF is at assessing PPIs of all kinds of size ranges.

Download Application Note

HTRF is suitable for low to high affinity interactions.

Protein-Protein interaction networks are extended and complex. They are also conveyed by a vast number of non-covalent binding modes. This diversity results in a wide range of affinities from very weak and transient interactions to very strong (High µM to picomolar).

The high sensitivity of HTRF, coupled with a wide diversity of PPI tools, enables the efficient measurement of complex interactions displaying low to high Kd.

HTRF PPI assays taken from published papers, displaying a large panel of affinities.

PPI assays, a large panel of affinities

Read this note to see how successful HTRF is at assessing PPIs with all kinds of affinity.

Download Application Note

Let’s see how to get started with your own PPI!

Custom make your own PPI
1

Choose your target interaction

Choose your target interaction

Receptor/ligands, RNA, DNA, lipids, enzymes, etc. Any interacting proteins are within your reach: It’s all about what you want!

2

Select assay format & starting material

Select assay format and starting raw material

Antibodies, native partner, biotinylated partner, tagged protein, Fc fused proteins matching your goals.

3

Choose your detection reagents

Choose your detection reagents

46 options ensure that you find the perfect match for your interaction: labeling kits, affinity reagents, detection and anti species reagents.

4

Assay optimization and data processing

Assay optimization and data processing

A single optimization step to adjust reagents to each other.
And off to the reader you go!

Get the essential steps

Read before you start the assay set up. Don’t do it alone, get the essential steps for developing your own PPI assay.

Download this technical note

Guidelines for optimizing protein-protein interaction assays using HTRF PPI reagents - Technical Note