Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions
Published: April 30, 2023
Abstract
Source: Wang, L. et al., Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay. J. Vis. Exp. (2021)
In this video, we demonstrate the amplified luminescent proximity homogeneous assay—a bead-based proximity assay. It utilizes homogeneously-sized acceptor and donor beads, immobilized with two proteins that tend to interact to screen potential small molecules inhibiting protein interactions.
Protocol
NOTE: An overview of this protocol is shown in Figure 1. 1. Expression and purification of GST-FKBP51 and GST-FKBP52 (Figure 1A) Plasmids NOTE: Obtain cDNA clones for human FKBP51 (clone id: 5723416) and for human FKBP52 (clone id: 7474554) from IMAGE consortium. Amplify the human FKBP51 DNA by PCR with primers (forward; 5`GGATCCATGACTACTGATGAAGGT-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing BamHI and …
Representative Results
Figure 1: Schematic of this protocol. (A) Expression and purification of GST-FKBP51 and GST-FKBP52. (B) Coupling of Hsp90 C-terminal peptide to the acceptor beads. (C) The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide. Inhibition with small molecular mass compounds.
Declarações
The authors have nothing to disclose.
Materials
| 384-well plates | Perkin Elmer | 6008350 | Assay volume 25 ml |
| Amicon 10.000 MWCO centrifugation unit | Millipore | UFC901008 | Concentrate protein |
| Ampicillin | Sigma | A0166 | Antibiotics |
| Bacteria shaker Unimax 1010 | Heidolph | Culture bacteria | |
| cDNA clones for human FKBP51 | Source BioScience | clone id: 5723416 | pCMV-SPORT6 vector |
| cDNA clones for human FKBP52 | Source BioScience | clone id: 7474554 | pCMV-SPORT6 vector |
| Chemically Competent E. coli | Invitrogen | C602003 | One Shot BL21 Star (DE3) |
| DMSO | Supelco | 1.02952.1000 | Dilute compounds |
| DPBS | Gibco | 14190-144 | Prepare solution |
| EDTA | Calbiochem | 344504 | Prevent proteolysis during sonication |
| Glutathione | Sigma | G-4251 | Elute GST-tagged proteins |
| Glutathione donor beads | Perkin Elmer | 6765300 | Donor bead |
| GST-trap column | Cytiva (GE Healthcare) | 17528201 | Purify GST-tagged proteins |
| Isopropyl-β-D-thiogalactoside | Thermo Fisher Scientific | R0392 | Induce protein expression |
| LB Broth (Miller) | Sigma | L3522 | Microbial growth medium |
| PCR instrument | BIO-RAD | S1000 Thermal Cycler | Amplification/PCR |
| PD-10 column | Cytiva (GE Healthcare) | 17085101 | Solution exchange |
| pGEX-6P-1 vector | Cytiva (GE Healthcare) | 28954648 | Plasmid |
| pGEX-6P-2 vector | Cytiva (GE Healthcare) | 28954650 | Plasmid |
| Plate reader | Perkin Elmer | EnSpire 2300 Multilabel Reader | Read alpha plate |
| Plate reader software | Perkin Elmer | EnSpire Manager | Plate reader software |
| Plate reader software protocol | Perkin Elmer | Alpha 384-well Low volume | Use this protocol to read plate |
| PMSF | Sigma | P7626 | Prevent proteolysis during sonication |
| protease inhibitor cocktail | Sigma | S8830 | Prevent proteolysis during sonication |
| Sodium azide | Sigma | S2002 | As a preservative |
| Sodium cyanoborohydride (NaBH3CN) | Sigma | 156159 | Activates matrix for coupling |
| Ten amino acid peptide NH2-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform | Peptide 2.0 inc | Synthesize Hsp90 C-terminal peptide | |
| Test-Tube Rotator | LABINCO | Make end-over-end agitation | |
| Tris-HCl | Sigma | 10708976001 | Block unreacted sites of acceptor beads |
| Tween-20 | Sigma | P1379 | Prevent beads aggregation |
| Ultra centrifuge Avanti J-20 XP | Beckman Coulter | Centrifuge to get bacteria cell pellets | |
| Ultrasonic cell disruptor | Microson | Sonicate cells to release protein | |
| Unconjugated acceptor beads | Perkin Elmer | 6762003 | Acceptor beads |
| XCell SureLock Mini-Cell and XCell II Blot Module | Invitrogen | EI0002 | SDS-PAGE |
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Citar este artigo
Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21231, doi: (2023).