Amersham rgb imager

The molecular size of the synthesized proteins was estimated using a SDS-PAGE and associated autoradiography. For this, 3 µL of the synthesized proteins were sampled and precipitated by the addition of ice-cold acetone (Carl Roth GmbH). After 15 min of incubation, samples were centrifuged at 16000g, 10 min, 4 °C to separate the precipitated protein and subsequently, air-dried. The protein pellet was resuspended in LDS sample buffer (ThermoFisher Scientific). After loading the sample on a precast Novex SDS-Gels (ThermoFisher Scientific), samples were run for 55 min. at 160 V at room temperature, stained (SimplyBlue Safe Stain, Life Technologies) dried at 70 °C on Whatman paper (Unigeldryer 3545D, Uniequip) and incubated in a phosphor screen for several days (GE Healthcare). The radiolabeled protein was visualized using a phosphor imager system (Amersham RGB Imager, GE Healthcare). The densitometric analysis of the complex formation was performed using ImageQuant TL software (TL 8.1- GE; Healthcare Bio-Science).

Saturation BRET experiments were performed constantly using 20 nM BR1-NLuc/Adora-NLuc and increasing BR2/BR2-mCitrine plasmid concentrations (0–50 nM) for CFPS. A 1:50 dilution of Nano-Glo^® luciferase substrate (Promega GmbH) to 5 µL of the VF was added. The assay was performed using a white plate in a Mithras² LB 943 multimode reader (Berthold Technologies). Raw values were measured and calculated as emission intensity at 530 nm divided by emission intensity using the OD2 filter: $${\mathit{BRET}}_{\mathit{ratio}}=\frac{Fluorescence(sample)}{Luminescence(sample)}-\frac{Fluorescence\left(c,o,n,t,r,o,l\right)}{Luminescence\left(c,o,n,t,r,o,l\right)}.$$
The BRET ratio was calculated as the ratio of a coexpressed sample with receptor and donor tag subtracted from the raw value obtained by coexpressing the Nluc-labelled receptor and the corresponding unlabelled receptor. The BRET ratio was calculated from three independent experiments (n = 3) and mean values and standard deviation were calculated and plotted.
The corresponding fluorescence detection was performed using 18-well μ-slides (IBIDI GmbH). Samples were transferred and at the excitation wavelength 488 nm using an Amersham RGB Imager (GE Healthcare).

5 µL aliquots of SN1 or SN2 were mixed with 45 µL ddH₂O and 150 µL pre-cooled acetone. Samples were incubated for a minimum of 15 min on ice and subsequently centrifuged at 16,000 × g and 4°C for 10 min. The resulting supernatant was discarded and the protein pellet was dried for at least 30 min at 45°C and resuspended in 20 µL 1x LDS sample buffer (NuPAGE™ LDS sample buffer (f.c. 1x), Thermo Fisher Scientific, Waltham, United States) supplemented with 50 mM (f.c.) DTT (Roche). Samples were heated for 10 min at 70°C and analyzed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) using self-made 14% Tris-Glycin gels (SureCast Gel Handcast System, Thermo Fisher Scientific, Waltham, United States) and Tris-Glycin running buffer. The electrophoretic separation was conducted at 155 V for 65 min. SDS-PAGE gels were subsequently stained using Coomassie blue solution (SimplyBlue SafeStain, Thermo Fisher Scientific, Waltham, United States) and dried on Whatman paper for 70 min at 70 °C (Unigeldryer, 3545D, Uniequip Laborgerätebau-und Vertriebs GmbH, Planegg, Germany). ¹⁴C-leucine-labeled protein bands were visualized using the Amersham RGB Imager (GE healthcare) after incubation of dried gels for at least three days on a phosphor screen (GE healthcare).