NOTE: This protocol for detergent screening is detailed for 30 g of HEK293 cell pellet as starting material.

1. Materials, chemicals and reagents

NOTE: All solutions are prepared using analytical grade reagents and ultrapure water, which is purified from deionized water to reach a resistivity of 18.2 MΩ∙cm at 25 °C.

1. Buffer stock solutions
   1. Prepare 10x phosphate buffered saline (10x PBS).
   2. Prepare HEPES buffer: 1 M, titrated to pH 7.5 with NaOH.
   3. Prepare 5 M NaCl.
   4. Prepare 2 M MgCl₂.
      NOTE: All stock solutions are passed through a 0.22 μm filter to maintain their sterility.
2. Detergent stock solutions
   1. Prepare dodecyl maltoside (DDM), 10% (w/v).
   2. Prepare decyl maltoside (DM), 10% (w/v).
   3. Prepare 6-cyclohexyl-hexyl maltoside (Cymal-6), 10% (w/v).
   4. Prepare 5-cyclohexyl-pentyl maltoside (Cymal-5), 10% (w/v).
   5. Prepare nonyl glucoside (C9G), 10% (w/v).
   6. Prepare lauryl maltose neopentyl glycol (LMNG), 5% (w/v).
   7. Prepare decyl maltose neopentyl glycol (DMNG), 10% (w/v).
   8. Prepare cymal-6 neopentyl glycol (C6NG), 10% (w/v).
   9. Prepare cymal-5 neopentyl glycol (C5NG), 10% (w/v).
   10. Prepare octyl glucose neopentyl glycol (OGNG), 10% (w/v).
       NOTE: For 10% detergent stock solution, dissolve 1 g of detergent powder in ultrapure water with gentle rocking, and then adjust the final volume to 10 mL. Detergent stock solution should be kept at -20 °C for long-term storage and on ice whilst working.
       CAUTION: Bottled detergents are usually recommended to store at -20 °C freezer. The bottles containing detergent powder should be warmed to room temperature before opening. Detergent powder is hygroscopic, so temperature equilibration will prevent the formation of condensation that will wet the detergent.
3. Other chemicals and reagents
   1. Prepare 1D4 immunoaffinity agarose resin: 10 mL of the 50% slurry.
      NOTE: The 1D4 immunoaffinity agarose are the agarose beads linked with the monoclonal Rho1D4 antibody, which binds the last 9 amino acids of bovine rhodopsin TETSQVAPA as epitope. The 1D4 immunoaffinity agarose works as affinity purification material to capture proteins that contain a C-terminal 1D4 sequence. This purification material can be prepared^9,11 or purchased.
   2. Prepare 9-cis retinal solution: 1 mM, dissolved in 100% ethanol.
      NOTE: Prevent light exposure to retinal during preparation and storage.
   3. Prepare 1D4 peptide (sequence TETSQVAPA): 800 μM, dissolved in water.
4. Buffers
   NOTE: All buffers are mixed from the stock solutions to the desired concentration. All buffers are chilled to 4 °C before use.
   1. Prepare Buffer A: PBS, 0.04% DDM.
   2. Prepare Buffer B: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.04% DDM.
   3. Prepare Buffer C: 20 mM HEPES pH 7.5, 150 mM NaCl, and detergent at their working concentration listed in Table 1.
   4. Prepare Buffer D: 20 mM HEPES pH 7.5, 150 mM NaCl.
   5. Prepare Elution buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 80 μM 1D4 peptide, and detergent at their working concentration.
   6. Prepare SEC buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.025% DDM; filtrated through a 0.22 μm filter.
5. Solvent for LC-MS
   1. Prepare solvent A: acetonitrile containing 0.1% formic acid.
   2. Prepare solvent B: ultrapure water containing 0.1% formic acid.
   3. Prepare solvent C: iso-propanol.

2. Cell membrane solubilization and protein extraction

1. Thaw 30 g of HEK293 GnTI^– cell pellet expressing the bovine rhodopsin mutant N2C/M257Y/D282C^3,9 to room temperature, add 120 mL of 1x PBS buffer containing protease inhibitor cocktail and homogenize using a Dounce homogenizer or an electric homogenizer (13,000 rpm for 30 s). Collect the homogenized cell suspension in a beaker and adjust the volume to 150 mL.
   NOTE: 30 g of cell pellet is equivalent to 3 L of cell culture at 2 x 10⁶ cell/mL density.
2. Gently add 10% DDM to the homogenized cells to give a final concentration of 1.25%. Stir on ice for 1 h.
3. Centrifuge the cell lysate at 4 ˚C and 150,000 x g for 45 min to remove the unsolubilized debris.
4. Transfer the supernatant to a 500 mL bottle and add 10 mL of the 1D4 immunoaffinity agarose resin (50% slurry). Gently mix the solubilized cell lysate and resin for 4 h or overnight at 4 °C.
5. Load the lysate/resin mixture to an open column to collect the resin.
6. Wash the resin with 10 column volumes (CV) of the wash Buffer A.
   NOTE: The column volume is the volume of the packed (100%) agarose resin used. In this case, 1 CV is 5 mL.
7. Resuspend the resin with 2 CV of Buffer A.
   CAUTION: From step 2.8 onwards, steps that need to be carried out under dim red-light condition are labelled with "[Dark]" at the beginning of the description.
8. [Dark] Add 9-cis retinal to the resuspended resin to the final concentration of 50 μM. Gently mix at 4 °C for 4-16 h in the dark.
   NOTE: A shorter incubation time may lead to incomplete reconstitution of retinal.
9. [Dark] Remove flow through from the column. Wash resin with 20 CV Buffer A, followed by 15 CV Buffer B.
10. [Dark] Resuspend the resin in 2 CV Buffer B, and then divide the resin suspension equally to 10 10-mL disposal columns.
11. [Dark] Remove flow through from the column, and then resuspend the resin in 1 mL Buffer C. Incubate for 1 h at 4 °C.
12. [Dark] Repeat step 2.11.
13. [Dark] Remove flow through from the column, and then resuspend the resin in 0.8 mL Elution Buffer for each column. Gently mix for 2 h.
14. [Dark] Collect elution from the column into a 2 mL tube.
15. [Dark] Resuspend the resin in 0.7 mL of Elution Buffer for each column. Gently mix for 1 h.
16. [Dark] Collect elution from the column into the same tube.

3. UV-VIS spectroscopy

1. Prepare the spectrophotometer to cover the measurement range of 250-650 nm. Record the baseline using water or Elution Buffer.
2. [Dark] Load the eluted protein to the quartz cuvette. Measure the spectrum of the protein sample.
3. [Dark] Illuminate the protein directly in the cuvette for 2 min with light passed through a 495 nm long-pass filter.
4. Measure the spectrum of the illuminated sample.
5. Perform the same measurement for all the protein samples purified in the other 9 detergents, both dark and illuminated states.
6. Plot the curves (absorbance versus wavelength) in X-Y scatter chart.

4. Automated size-exclusion chromatography of rhodopsin and rhodopsin–mini-G_o complex

1. [Dark] Concentrate protein to 100 μL by centrifugation using a spin concentrator with a molecular weight cut-off (MWCO) of 30 kDa at 4 °C. Overconcentrated samples can be diluted using the flow through from the concentrator or Buffer C. To determine protein sample concentration, measure absorbance at 280 nm using a spectrophotometer.
   NOTE: From step 4.2 onwards, the experiment does not require a dark environment, and therefore samples can be prepared under normal light.
2. Prepare 100 μL rhodopsin at 0.7 mg/mL for each detergent condition.
3. Prepare 100 μL of rhodopsin (0.7 mg/mL) and mini-G_o^4,12 (0.2 mg/mL) mixture for each detergent condition. Supplement the mixture with 1 mM MgCl₂. Illuminate the mixture with light from a 495 nm long-pass filter and incubate for 30 min.
4. Mount a 24 mL gel filtration column with a fractionation range of 10-600 kDa of a globular protein on a liquid chromatography purifier. Equilibrated the column with SEC buffer.
   NOTE: The liquid chromatography purifier is equipped with an autosampler, a multiple wavelength detector and a fraction collector.
5. Transfer the samples to the autosampler vials and place them in the sample tray. Program a method file to automate sequential SEC runs for each sample, with the autosampler loading 77 μL of the sample to the column, and the purifier eluting 24 mL of SEC buffer at a flow rate of 0.5 mL/min per run. Record the absorbance at 280 nm and 380 nm.
6. Collect the peak fractions of rhodopsin and rhodopsin–mini-G_o complex at the retention volume around 12.9 mL.
7. Analyze the left rhodopsin samples from step 4.2 and the peak fractions of rhodopsin–mini-G_o complex on 4-12% SDS-denaturing gradient gels with Coomassie blue staining.
8. Plot the elution chromatogram (A₂₈₀ or A₃₈₀ versus retention volume).

5. Deglycosylation and LC-MS study

1. For LC-MS study, only use the rhodopsin sample purified in LMNG detergent.
2. Prepare a 200 μL mixture of rhodopsin at 1 mg/mL and PNGase F¹³ at 0.01 mg/mL. Mix well and incubate at 4 °C overnight.
3. Prepare a 200 μL mixture of rhodopsin at 1 mg/mL and Endo F1¹³ at 0.01 mg/mL. Mix well and incubate at 4 °C overnight.
4. Analyze the digestion result by SDS-PAGE and Coomassie blue staining.
5. Concentrate untreated and Endo F1-treated rhodopsin samples and subject to SEC purification in Buffer D.
   NOTE: This is to prepare the sample with minimal quantity of detergent for LC-MS study. Buffer D does not contain any detergent, but due to the slow off-rate of LMNG from a membrane protein¹⁴, rhodopsin will not aggregate.
6. Collect the peak fraction at retention volume around 12.9 mL. Concentrate to 1 mg/mL using a spin concentrator (MWCO 30 kDa).
7. Inject 10 μg of the protein into a Reprosil 200 C18-AQ column and elute the column using the linear gradient method with the solvent composition and settings listed in Table 2. The flow is split to 25% for mass spectrometer and 75% for UV detection.