FRET Characterization of BMPR1 and Iβ1 Interaction

To investigate the association of BMPR1 and Iβ1, we performed Förster Resonance Energy Transfer (FRET)^{23 (link), 27 (link)}. Briefly, the donor antibody (anti Iβ1 (SantaCruz)) was labeled with Alexa Fluor^® 488 Carboxylic Acid, Succinimidyl Ester (Life Technologies) (Iβ1-488) and the acceptor antibody (anti TGF-β RII (SantaCruz)) was labeled with Alexa Fluor^® 546 Carboxylic Acid, Succinimidyl Ester (Life Technologies) (BMPRI-546) in a 2.25:1 molar ratio of antibody:dye overnight at 4°C. Labelled antibody was purified with size exclusion chromatography using PD-10 Desalting Columns (GE Healthcare). Rat MSCs were plated on pre-soaked compliant (5 MPa) and rigid (266 MPa) PUR films. After 24 hours of culture, cells were fixed with 10% Formaldehyde in PBS and stained overnight at 4° with Iβ1-488 and Iβ1-488 + BMPRI-546 (1 ug/1×10⁶ cells). FRET experiments were performed on a BioTek Synergy 2 plate reader using excitation filter 485/20 and emission filter 530/35.

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Guo R., Lu S., Merkel A.R., Sterling J.A, & Guelcher S.A. (2016). Substrate Modulus Regulates Osteogenic Differentiation of Rat Mesenchymal Stem Cells through Integrin β1 and BMP Receptor Type IA. Journal of materials chemistry. B, Materials for biology and medicine, 4(20), 3584-3593.

Publication 2016

anti TGF-β RII Alexa Fluor® 546 Carboxylic Acid, Succinimidyl Ester PD-10 Desalting Columns Synergy 2 plate reader

Alexa fluor 488 Alexa fluor 546 Antibody Carboxylic acid Cells Donor Ester Formaldehyde Förster resonance energy transfer Molar Rigid Size exclusion chromatography Tgf β

Corresponding Organization : Colorado Center for Bone Research

Other organizations : Nashville Oncology Associates, VA Tennessee Valley Healthcare System

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Variable analysis

independent variables

Substrate stiffness (compliant 5 MPa vs rigid 266 MPa PUR films)

dependent variables

FRET signal between Iβ1-488 and BMPRI-546

control variables

Rat MSCs
Culture duration (24 hours)
Antibody concentrations (1 ug/1x10^6 cells)

controls

Positive control: Iβ1-488 (Iβ1 labelled with Alexa Fluor 488)
Negative control: Not mentioned

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