TITLE: Analysis of De Novo Synthesized Proteins
AUTHORS: Anne Zemella, Theresa Richter, Lena Thoring, Stefan Kubick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This is part 3.4 of the "A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties" collection of protocols</span><span>: </span><a href="https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmve</span></a><a href="https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;font-weight:bold;">n</span></a></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET).</span></div><br/><div class = "text-block"><span>For </span><span style = "font-weight:bold;">Introduction and Notes</span><span>, please see: </span><a href="https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines</span></a></div></div>

[STEPS]
?. [3.4.1 TCA Precipitation and Scintillation Counting]
After the reaction is completed collect 2 × . Centrifuge the remaining mix at  and collect 2 × . Resuspend the microsomal fraction in an equal volume of PBS in comparison to the volume of the translation mixture. Collect 2 × .
[translation mixture]
Centrifuge: 16000 34, 15 min, 4 10
[supernatant]
[microsomal fraction]
?. [3.4.1 TCA Precipitation and Scintillation Counting]
Mix each aliquot with  and incubate in a water bath at  for . Store the aliquots for  or  at .
[TCA]
80 °C
30 °C
on ice
4 °C
?. [3.4.1 TCA Precipitation and Scintillation Counting]
The mixture is applied to a vacuum filtration system to separate non-incorporated 14C-leucine from the radioactively labeled protein. Filters with the collected protein are washed twice with TCA and twice with acetone. Dry the filters under the hood.
?. [3.4.1 TCA Precipitation and Scintillation Counting]
The filters are transferred into the scintillation vials and overlaid with . After an incubation time of  with gentle shaking, scintillation vessels are counted in scintillation counter.
[scintillation cocktail]
?. [3.4.2 In-Gel- Fluorescence and Autoradiography]
For preparation of SDS-PAGE samples take a  (part 3.3, section 3.3.1 "Fluorescent Labeling with Bodipy-TMRLysine", steps 3–5) or  (part 3.3, section 3.3.3 "Site-Specific Incorporation of a Non-canonical Amino Acid with Subsequent Fluorescent Labeling and Microscopic Analysis", steps 13–17) of each prepared sample.
[aliquot]
[site-specifically labeled aliquot]
?. [3.4.2 In-Gel- Fluorescence and Autoradiography]
Dry the pellets for  at  in a thermo mixer with a shaking speed of .
45 °C
33
?. [3.4.2 In-Gel- Fluorescence and Autoradiography]
Resuspend the dried pellets in  and load the samples on a prepared . Use a ladder with fluorescently labeled bands. Run the gel.
[SDS-PAGE sample buffer]
[SDS-PAGE gel]
?. [3.4.2 In-Gel- Fluorescence and Autoradiography]
Transfer the gel to the fluorescence imaging system and detect the labeled protein bands. For Bodipy-TMR-lysine use a 532 nm laser and a 580 nm emission filter. Sulfo-Cy5 can be detected with extinction at 633 nm and emission at 670 nm.
?. [3.4.2 In-Gel- Fluorescence and Autoradiography]
Afterwards dry the gel for  at  using a unigeldryer. The dried gels are exposed on a phosphorscreen for minimal 3 days and read out using a multi-mode imager.
70 °C
?. [3.4.3 Confocal Laser Scanning Microscopy]
For confocal laser scanning microscopy use  in the microsomal fraction and dilute the sample in . Add the mixture to a μ-Ibidi-Slide.
[fluorescently labeled protein]
[PBS]
?. [3.4.3 Confocal Laser Scanning Microscopy]
Fix the slide. Use a plan-apochromat objective with a 60× or 100× magnification. Microsomal structures usually have a diameter of 1–10 μm.
?. [3.4.3 Confocal Laser Scanning Microscopy]
Adjust the beam path to the coupled fluorescent dye. Standard dyes such as Cy5 and FITC usually have a preset configuration. Cy5 is excited at 633 nm and the emission is detected with a long-pass filter above a wavelength of 670 nm.
?. [3.4.3 Confocal Laser Scanning Microscopy]
Adjust the microscope settings (laser intensity, gain master, focus, pinhole) according to the individual sample (see Note 17).
?. [3.4.2 In-Gel- Fluorescence and Autoradiography]
Add  and  to the  or  and incubate for . Keep the fluorescently labeled samples in dark during the whole procedure. Centrifuge the samples at  and discard the supernatant.
[water]
[cold acetone]
5 µl
[aliquots]
on ice
Centrifuge: 16000 34, 10 min, 4 10