Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization

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

1. 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.
2. Transfer transformed colonies into 100 mL LB starter culture and grow for 16 - 20 hours at 37 °C with shaking.
3. Dilute dense starter culture (OD600 >1) 1:100 in LB medium (2 x 500 mL batches) and grow for 2 - 3 hours to OD600 = 0.6 - 0.8 (37 °C, 200 rpm ).
4. Cool cultures on ice. Induce with 250 µM IPTG and grow for ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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