Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization
Published: May 31, 2023
Abstract
Source: Nolan, R., et al. Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software. J. Vis. Exp. (2018)
This video demonstrates the technique of fluorescence fluctuation spectroscopy (FFS) to study protein homo-oligomerization. Fluorescently-labeled proteins in a sample are dimerized using an agent when studying protein oligomerization using FFS. Using a confocal microscope, as the proteins move in and out of the small observation volume, the analysis of the fluctuations in the brightness of the fluorescent molecules is performed to determine the oligomeric state of the proteins.
Protocol
1. FKBP12 F36V -mVenus Purification Transform (DE3) pLysS cells with pET22b vector containing monomerized human FKBP12F36V12 and N-terminal His6 and mVenus tags (vector available on request). Plate cells onto LB agar supplemented with 50 µg/mL Ampicillin and 34 µg/mL Chloramphenicol. Transfer transformed colonies into 100 mL LB starter culture and grow for 16 – 20 hours at 37 °C with shaking. Dilute dense starter culture (OD600 >1) 1:100 in LB medium (2 x 500 mL batc…
Representative Results
Figure 1. Application of N&B to detect protein monomer-dimer transitions in solution. (a) Simplified optical path of a laser scanning microscope (LSM) equipped with a laser source (set at 514 nm in the case of mVenus labeled proteins) directed (blue arrows) toward an immersion objective (in our case a 63X1.4NA oil) illuminating a 100 nM solution of FKBP12F36V-mVenus solution. The emission flu…
Disclosures
The authors have nothing to disclose.
Materials
| RosettaTM (DE3) pLysS cells | Novagen | 70956-3 | |
| Ampicillin | Sigma Aldrich | PubChem Substance ID 329824407 | |
| Chloramphenicol | Sigma Aldrich | PubChem Substance ID: 24892250 | |
| LB starter culture | QIAGEN | ||
| LB medium | QIAGEN | https://www.sigmaaldrich.com/content/dam/sigma-aldrich/head/search/external-link-icon.gif | |
| IPTG | Sigma Aldrich | PubChem Substance ID 329815691 | |
| IMAC buffer | Medicago | 09-1010-10 | |
| EDTA-free protease inhibitors | Sigma Aldrich | 11873580001 | |
| TALON resin | Clonetech | ||
| Nickel sepharose | GE Healthcare | ||
| S200 16/60 column | GE Healthcare | ||
| Glass bottom 8-well observation dish | Ibidi | 80827 |
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Cite This Article
Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization. J. Vis. Exp. (Pending Publication), e21409, doi: (2023).