- freely available
Methods Protoc. 2019, 2(1), 6; https://doi.org/10.3390/mps2010006
2. Experimental Design
- Acetal–PEG27–NHS (available by purchase from H. Gruber, Johannes Kepler University Linz , Linz, Austria). Alternatively, this linker can be synthesized according to Reference . CRITICAL STEP We recommend purchasing monodisperse polyethylene glycol (PEG) linkers from this source for two reasons. First, the benzaldehyde function is protected by an acetal-group for the binding to amino-functionalized tips via the activated carboxyl functionality of the N-hydroxysuccinimide (NHS)-group. This elegantly precludes the possibility of bivalent interaction of the benzaldehyde function with adjacent amino-groups on the tip surface. Second, the linker is supplied in high-purity 1-mg aliquots ideally suited for use in this protocol.
- Dimethylsulfoxide anhydrous (>99.9%, (CH3)2SO (DMSO); Sigma–Aldrich, St. Louis, MO, USA, cat. no. 41640) CAUTION DMSO is harmful upon inhalation and skin absorption. Wear appropriate gloves.
- Sodium cyanoborohydride (NaCNBH3; Sigma–Aldrich, cat. no. 156159) CAUTION NaCNBH3 is highly toxic. Wear gloves and handle carefully.
- Chloroform (>99.9%, CHCl3; Sigma–Aldrich, cat. no. 288306) CAUTION Chloroform is highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and work under a fume hood. Handle only in glass vessels and glass dishes covered with a lid.
- Ethanol, absolute (>99.9%, C2H5OH; Sigma–Aldrich, cat. no. 24102).
- (3-Aminopropyl)triethoxysilane (APTES, 99%, Sigma–Aldrich, cat. no. 440140). Distill at low temperature and store under argon in sealed crimp vials over silica gel at −20 °C to avoid polymerization.
- Ethanolamine hydrochloride (H2NC2H4OH; Sigma–Aldrich, cat. no. E6133).
- 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617).
- Molecular sieves, 3 Å (beads, 8–12 mesh; Sigma–Aldrich, cat. no. 208574).
- Triethylamine >99.5%, (C2H5)3N; Sigma–Aldrich, cat. no. 471283) CAUTION Triethylamine is harmful upon inhalation and skin absorption, and is volatile. Wear appropriate gloves and work under a fume hood. Store under argon and in the dark to avoid amine oxidation.
- Milli-Q water (Merck Millipore, Burlington, MA, USA).
- Citric acid (Sigma–Aldrich, cat. no. 251275).
- Stock of membrane proteins, reconstituted into proteoliposomes. UCP1 and SecYEG for the examples shown here.
- Ethylenediamine (EDA) derivates of the purine nucleotides (EDA-ATP, EDA-ADP and EDA-AMP) (BioLog, Bremen, Germany, cat. no. A072, A118) or SecA protein for tip coupling.
- Na2SO4 (Sigma-Aldrich, cat. no. 238597).
- (NH4)2HPO4 (Sigma–Aldrich, cat. no. 215996).
- 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma–Aldrich, cat. no. M3671).
- Tris (Sigma–Aldrich, cat. no. 252859).
- Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma–Aldrich, cat. no. E3889).
- HEPES (Sigma–Aldrich, cat. no. H3375).
- HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527).
- Sodium Chloride (NaCl) (Sigma–Aldrich, cat. no. S7653).
- Calcium Chloride (CaCl2) (Sigma–Aldrich, cat. no. C1016).
- Magnesium Chloride (MgCl2) (Sigma–Aldrich, cat. no. M8266)
- Potassium Chloride (KCl) (Sigma–Aldrich, cat. no. P9333)
- Muscovite mica sheets (V-1 quality, Electron Microscopy Science, Hatfield, PA, USA, cat. no. 71855)
- Keysight 5500 AFM setup (or higher) equipped with PicoPlus box.
- Closed-loop scanner (N9524A, Keysight).
- AFM tapping nose cone for acoustic excitation (Keysight).
- MSNL-lever (Bruker AFM Probes).
- Inverted optical microscope.
- Upright benchtop optical microscope for examining cantilevers and chips.
- Active vibration isolation table.
- Acoustic enclosure for the AFM.
- AFM flow-through liquid cell.
- N2 gas bottle for drying cantilever chips.
- Argon gas bottle for storing cantilever chips and chemicals properly without contamination.
- pH meter.
- Glass Pasteur pipettes and rubber bulbs for pH adjustment.
- Analytical balance.
- Fume hood.
- Vacuum pump.
- Vacuum desiccator.
- Plastic and glass Petri dishes (15 mm) with lid for tip functionalization and surface chemistry.
- Mechanical pipettors.
- Pipette tips.
- Hot plate stirrer.
- Magnetic stir bars.
- Glass beakers and Erlenmeyer flasks.
- 4-cm glass crystallization dishes (Sigma–Aldrich, cat. no. BR455701).
- Teflon block (3 × 1 × 0.5 cm).
- Teflon reaction chamber (2 × 2 × 1.5 cm block indented with a cylindrical chamber 1-cm deep and 1.5 cm in diameter).
- 12-well plates (Sigma–Aldrich, cat. no. CLS3513).
- 50 µL and 500 µL Hamilton glass syringe.
- Gwyddion Analysis software (v. 2.49) or any other analysis software for scanning probe microscopy (SPM) images, such as Pico Image (Keysight) or ImageJ.
- Origin (Origin 2016, Origin) or any other scientific graphing and data analysis software.
- Matlab (2014 or higher) + kspec19 analysis routine (provided by Keysight).
3.1. Proteoliposome Preparation and Protein Reconstitution. Time to Completion: Several Days, Depending on the Studied Biomolecule and Applied Protocol for Preparation
3.2. Surface Chemistry. Time to Completion: 15 min up to ~2 h, Depending on the Studied Biomolecule
- Prepare a freshly cleaved mica by removing the upper layer of the quartz-like material with a commercial tape.CRITICAL STEP Make sure to remove a closed layer, to generate an ultra-flat and clean surface for incubation of the protein-containing proteoliposomes.
- Place the freshly cleaved mica on the AFM sample plate and mount a (flow-through) fluid cell.
- Dilute the proteoliposome solution to a final concentration of 1 mg/mL lipid concentration with assay buffer.
- After short vortexing, pipet the proteoliposome solution into the fluid cell and onto the clean and flat mica sheet.
- Depending on the lipid composition and the protein, let the sample incubate for 10 min up to 2 h at room temperature (RT). After that, lipid bilayer batches containing the protein of choice should be formed.OPTIONAL STEP Depending on the lipid composition (and their phase transition temperatures), you may raise the temperature up to 60 °C during the incubation.CRITICAL STEP Make sure to always keep the sample moist to prevent evaporation of the liquid during the incubation.
- Wash the sample thoroughly with assay buffer and add 500–600 µL assay buffer to the fluid cell for AFM measurements.
3.3. AFM Tip Functionalization. Time to Completion: 2–3 Days, Depending on the Chosen Aminofunctionalization Procedure
3.3.1. Aminofunctionalization of the AFM Tip
- CRITICAL STEP Perform the whole procedure in a well-ventilated hood.
- Wash cantilevers in chloroform (3 × 5 min), dry with nitrogen gas, continue with the next step.
- Flush desiccator chamber (5 L) with argon gas (through the narrow opening in the lid).
- Place a tray (caps from 1.5 mL plastic Eppendorf tubes) with 30 μL APTES and another tray with 10 μL triethylamine in the desiccator.
- Place the cantilevers in the desiccator close to the two trays. Close the lid, incubate for 2 h.
- Remove the trays with APTES and triethylamine, flush desiccator with argon. Incubate the tips in argon for at least 2 days (“curing”).
- PAUSE STEP Store under argon in a dust box for up to 3 weeks (preferably <1 week), if they are not used immediately.
- Wash cantilevers in chloroform (3 × 5 min), dry with nitrogen gas, continue with the next step.
- Dissolve 3.3 g ethanolamine hydrochloride in 6.6 mL DMSO, cover with lid, heat to ~70 °C for complete dissolution, let cool to room temperature. In addition, a magnetic stirrer can be added to the solution to speed up the dissolving process.
- Immerse a Teflon block for the cantilevers in the center of the dish and add 3–4 Å molecular sieves beads to cover the surrounding area (~25% of the total volume of the liquid).
- Put the dish into a desiccator (or vacuum chamber) and degas the solution and the molecular sieves by applying an aspirator vacuum (or similar) for ~30 min.
- Place cantilevers on the Teflon block, cover with lid, incubate at room temperature overnight.
- Wash cantilevers in DMSO (3 × 1 min) and ethanol (3 × 1 min).
- Dry with a gentle stream of nitrogen or argon gas.
- PAUSE STEP Store under argon in a dust box for up to 3 weeks (preferably <1 week), if they are not used immediately.
3.3.2. NHS–PEG27–Acetal Coupling
- Dissolve 1 portion of Acetal–PEG27–NHS (1 mg) in chloroform (0.5 mL), transfer the solution into the reaction chamber, add triethylamine (30 μL) and mix by pipetting.
- Immediately place cantilever(s) in the reaction chamber, cover the chamber and incubate for 2 h.
- Wash with chloroform (3 × 10 min) and dry with nitrogen or argon gas.
- PAUSE STEP Store cantilever(s) for up to several months under argon, or continue with next step (Section 3.3.3).
3.3.3. Coupling of Interacting, Corresponding Biomolecule to the Free Acetal-End
- Immerse the cantilevers functionalized with NHS–PEG27–Acetal cross linker for 10 min in 1% (w/v) citric acid (in water).
- Wash the cantilevers in Milli-Q water (3 × 5 min) and dry with nitrogen or argon gas.
- Freshly prepare a 1 M solution of sodium cyanoborohydride (with 20 mM NaOH) from the following components: 13 mg NaCNBH3, 20 μL 100 mM NaOH, and 180 μL Milli-Q water.CRITICAL STEP NaCNBH3 is highly toxic. Wear gloves and handle with care.
- Place the AFM chips in a radial arrangement, with the tips in the center and facing upwards, on Parafilm in a polystyrene Petri dish.
- Pipet 100 μL protein solution (1–2 μM) carefully onto the tip of each cantilever so that they are extended into the protein drop. In the first example shown here, an EDA derivate of different purine nucleotides was coupled to the AFM tip to study their interaction with uncoupling proteins. In the second example, the ATP motor-protein SecA was attached onto the AFM tip in order to investigate its interaction with the bacterial translocon SecYEG.
- Add 2 μL of the freshly prepared 1 M NaCNBH3 stock solution to the protein solution droplet, mix carefully by pipetting, cover with a lid, and incubate for 1 h.
- OPTIONAL STEP Dissolve 3 pellets NaOH in 500 mL water, add the unused NaCNBH3 solution, mix, wait overnight, pour into the drain, and flush with tap water.
- To passivate unreached aldehyde groups, add 5 μL of ethanolamine solution (1 M, pH 8.0) to the drop on the cantilever(s), mix cautiously by pipetting, cover with a lid, and incubate for 10 min.
- Wash in PBS or any other buffer of choice (3 × 5 min).
- Mount cantilever in AFM setup if used immediately for experiments.PAUSE STEP If not used immediately, store cantilever in a 24-well plate under PBS at 4 °C for 1–2 weeks (depending on the stability of the conjugated biomolecule).CRITICAL STEP Transfer the functionalized AFM cantilevers to PBS buffer and then to the AFM always very rapidly so that they do not dry (within 20 s). Otherwise, the conjugated biomolecule could start to denaturate and lose its bio-functionality.
3.4. AFM Measurement. Time to Completion: ~4 h up to 1 Day, Depending on the Studied Biomolecule and the Desired Data Throughput
- Switch on the AFM equipped with a PicoPlus 5500 box (Keysight Technologies) and the AFM controlling software (PicoView, usable versions starting from 1.12 until 1.20).
- Switch on the inverted optical microscope and the related controlling software.
- Mount the AFM liquid cell with the sample on the AFM stage from the bottom.
- Mount the AFM tip derivatized with the corresponding, interacting molecule onto the cantilever holder (tapping nose cone), slot the holder into the closed-loop scanner and assemble everything onto the AFM stage from the top.CRITICAL STEP Ensure that the Z stepper motors are sufficiently retracted to prevent crashing the cantilever into the bottom to the fluid cell and onto the sample.CRITICAL STEP Perform these steps rapidly. A drop of the assay buffer should always remain on the cantilever chip during transfer and should not be allowed to dry out.CRITICAL STEP This experiment works only with closed-loop scanners (e.g., N952A, Keysight Technologies) and the tip acoustically excited. Make sure to connect the cable for the closed-loop scanner on the AFM stage and switch on the closed-loop option in the software. Moreover, use the right nose cone (for acoustic excitation, “tapping nose”).
- Align the laser spot at the free end of the cantilever and maximize the sum into the photodetector.
- Bring the cantilever close to contact with the sample as follows: Using a wide-field imaging mode and the manual controls, locate the shadow of the cantilever by adjusting the AFM head position until the cantilever is directly in the light path. Using the AFM software, slowly drive the Z stepper motors of the AFM head down until the cantilever is within the focal range of the microscope objective and bring it into focus. It might be necessary to make fine adjustments in the position of the laser spot at this time.
- Allow 15 min up to 1 h for the system to equilibrate, until the vertical offset of the laser spot on the photodiode no longer drifts. Try to keep the lab temperature constant during the whole experiment.CRITICAL STEP Perform this step very thoughtfully and considerate. A stable system with as less thermal drift (in x–y directions) as possible is very important for the upcoming experimental steps.
3.4.1. TREC Imaging: Protein Distribution and Functionality
- Choose AFM tapping mode in the software.
- Select the following channels for recognition imaging: height, recognition image channel (commonly referred as AUX channel), amplitude, and phase—both for trace and retrace.
- Run a tuning curve (0–50 kHz) to set a proper frequency for the acoustic excitation of the cantilever . To achieve an adequate driving amplitude, chose the second frequency level (f ~ 21 kHz) for recognition imaging instead of the base resonance frequency (f ~ 10 kHz) for the MSNL lever.
- To engage, choose the following settings:
- Amplitude setpoint ~4 V, adjust with drive.
- Stop at amplitude reduction of 80%.
- Start the imaging and optimize the AFM recognition imaging parameters (amplitude setpoint, oscillation frequency, feedback gains). This is partially a trial-and-error process and will differ between the samples, as well as the coupled biomolecule to the tip. Topographs should appear similar regardless of scanning direction (trace and retrace). The corresponding recognition events, displayed in the AUX channel, should match with topographic features. A brief description of some key parameters follows, along with suggested value ranges and can be found in more detail in Preiner et al. :
- The proper chosen oscillation amplitude is the most prerequisite to achieve reliable recognition events. If the linker is stretched too less at low oscillation amplitudes or, alternatively, if the receptor—ligand bond is broken at each oscillation cycle when using too large amplitudes, no pronounced and stable recognition events are observed. The range of appropriate amplitudes for recognition imaging is determined by the effective linker length (including the position and the length of the molecule attached to the linker), and therefore sharply localized. Run an amplitude–distance curve and test different amplitude setpoints (while keeping the ratio of amplitude setpoint and free amplitude constant) to find the best working amplitude for achieving pronounced recognition signals. The value varies usually between 1.5 and 2 V.
- Additionally, the variation of the amplitude can be also used as a specificity proof, besides the widely used tip/surface block (Figure 2b) without perturbation of the receptor–ligand system. This procedure is based on the physical property of the signal transduction of the recognition process. Vary the amplitude to very high (~3–4 V) and very low (<1 V) values. If the recognition spots disappear at this value and appear again at the proper set value (~2 V), it will give the user the proof for specific interaction events between the chosen receptor–ligand pair.
- The oscillation frequency has direct influence on the topographical crosstalk in the recognition signal. Adjust the value always lower than the resonance frequency (2nd order) of the cantilever at tip/surface contact. For lower driving frequencies, the topographical perturbation has more time to decay until the recognition signal is detected.
- Optimize the feedback gains to allow the AFM tip to accurately track the surface. Increase the gain to the point at which feedback oscillation noise is observed in the AFM topography image. Slightly reduce the gain to remove this effect.
- Scan the sample with above suggested parameters and find a good sample position, more precisely an area with single proteins incorporated in the lipid membrane and with clearly defined recognition spots which match with the topography. For doing so, first scan a bigger area (~5–10 µm) to find single, big bilayer batches and gradually zoom-in to visualize the proteins in the membrane and to study their recognition with the derivatized molecule on the tip (500–700 nm scan size).
- OPTIONAL STEP Change the sample and/or adjust the incubation time, lipid concentration, temperature, etc., to optimize the formation of single, big lipid bilayer batches on mica with the incorporated molecule homogenously distributed (Figure 2a). If no recognition signals appear after adjusting all parameters, change the tip.
- After finding a suitable area with a size of 500–700 nm2 that covered by a complete membrane batch check whether the membrane proteins are mostly visualized as single, round-shaped structures showing specific interaction (Figure 2a). Then start scanning this area several times up and down without changing any parameters. As soon as no thermal drift occurs anymore, and the subsequent scans show no change in terms of position displacement/shift, stop the measurement and switch from tapping to contact mode.CRITICAL STEP Before switching to contact mode, make sure that the thermal drift is as minimal as possible, since the subsequent force spectroscopy experiment is carried out “blindly” without a visual control of the correct cantilever position on the depicted molecule.
3.4.2. Force Spectroscopy: Dynamics and Kinetics of Interacting Partners
- After changing to contact mode, position the cantilever on a selected recognition spot (functional molecule showing strong binding in TREC mode) using positional feedback control.
- Start the force spectroscopy experiment by recording force distance curves directly on the depicted spot using well-adjusted parameters (pulling speed, hold times, etc.) (Figure 1c). Since binding/unbinding is a stochastic process record at least 500–1000 curves per pulling speed.
- OPTIONAL STEP In order to probe the dynamics and kinetics of the receptor-ligand interaction vary the pulling speed, and thus, loading rate after recording a data set of 500–1000 curves.
- CRITICAL STEP Thermal drift may occur during the force spectroscopy experiment and the user will lose the depicted protein while recording force distance curves. This can be recognized from a dramatic drop in the binding probability and no more characteristic binding events in the retraction curve. To readjust the cantilever on the depicted spot, switch back to tapping mode and repeat steps 6 and 8 from Section 3.4.1 to find/trace back the probed molecule. Do not chance the scanning parameters. Start again with steps 1–3.
- To calibrate the cantilever spring constant apply the thermal noise method according to Hutter et al.  at the end of the experiment. Repeat the spring constant determination for each used tip at least five times.
- Perform a specificity proof, in addition to the amplitude block experiment. After finding a proper scanning area with several recognition spots, inject a surface blocking molecule (~1–10 mM) and scan this area until the recognition spots disappear. Use an excess of blocking molecules (mM range) to guarantee that all the surface molecules are saturated. An almost complete loss of the recognition events after addition of the free molecule, while leaving the topography image unchanged (Figure 2c), will clearly confirm that most of the recognition spots were caused by specific binding of the tip-linked molecule to the proteins in the planar lipid membrane. Select a molecule where the recognition has disappeared and record at least one data set of force distance curves. You should detect almost no binding events with binding probabilities below 5%, due to the specific blocking of the ligands binding pocket in the receptor. Additionally, recording some force distance curves on the pure lipid membrane should also reveal little to no binding events, since specific interaction with the tip-linked ligand and the lipids is not expected. The few remaining recognition/unbinding events can be attributed to unspecific adhesion.
4. Expected Results
4.1. Proof-of-Concept: UCP-PN Interaction
4.3. Data Analysis
4.3.1. AFM Topography and Recognition Images
- Load the topography and recognition image into Gwyddion and visualize the in a false-color image (topography: e.g., “golden”; recognition: e.g., “grey scale” or any other color pattern to properly distinguish between topography and adhesion events).
- Improve the height images by using filters such as flatten or plane fit. CRITICAL STEP Users should keep in mind that application of any filter will—to some extent—modify the raw values and that quantitative parameters should be extracted before use of filters.
- In order to analyze the height and diameter of the (single) molecules, use a zoom-in image and extract cross-sectional profiles.
- For allocation of recognition events to topographical features, produce an overlay of those two channels.
- For comparing different amplitudes at the same scan area (amplitude block) or the images taken before/after the surface block, keep the z-scale value (to adjust the contrast), as well as the threshold (recognition yes or no) always constant. Determine the right threshold from regions without a protein; only when the recognition signal is below this value, consider the protein as to be recognized by the ligand functionalized tip.
4.3.2. Analysis of the Force Curves
- Load the recorded FD-curves into suitable scientific analysis software, such as Matlab (custom-written routines from Keysight commercially available), Gwyddion or PicoView.
- Depending on the used software, extract the following values:
- Loading rate from each rupture event: Measure the slope of the force versus distance curve just before the rupture. This gives the value of the effective spring constant keff (force segment ∆F divided through the distance segment ∆x of the very last part of the rupture event). Multiply this value with the applied pulling speed. Alternatively, as a more elegant way: If force vs. time curves were additionally recorded, the slope of the force vs. time curve just before the rupture event directly results in the loading rate.
- Rupture force: Take the value on the y-axis (force axis) right on the (negative) maximum peak of the rupture event before it returns to baseline level. In case of curves containing multiple rupture events, each rupture should be considered separately.
- Plot the extracted unbinding force versus the loading rate on a semi-logarithmic scale (DFS plot) using Origin or a similar software, which allows to extract kinetic and thermodynamic parameters using appropriate models and strategies .
- If desired, make unbinding force histograms or probability density functions (pdfs) from separated, distinct loading rate ranges to capture all the information contained in the DFS plot, such as the presence of multiple interactions. Fit the histograms with Gaussian distributions to determine the presence of single or multiple peaks, directly corresponding to one or several receptor–ligand interaction(s). The advantage of using pdfs instead of histograms is that data binning is avoided and the data will be analyzed more precisely, since the values are weighted to their reliability .
- Fit the loading-rate-dependent interaction forces with the fitting model of choice. Such models can be a linear iterative fitting algorithm (Levenberg Marquardt) along with the Bell–Evans model  (Figure 3a) or a non-linear iterative fitting algorithm (Levenberg Marquardt) along with the Friddle–Noy–de-Yoreo (FNdY) model . The simplest and most commonly used model is the Bell–Evans fit for crossing a single energy barrier, where the user is obliged just to take single interactions into account. Here, exclude multiple binding events from the force pdfs, which appear as a “shoulder” in the Gaussian distribution of the most probable unbinding force. Exclude these data and use only data within the interval [μ ± σ] for construction of the DFS plots and the subsequent fitting with the Bell–Evans model.
- An extensive overview of analysis and fitting of dynamic force spectroscopy (DFS) data is given by Bizzarri and Cannistraro , as well as by Noy and Friddle , whereas Hane and colleagues have compared the dominant models in great detail . The choice of model for data analysis relies first and foremost on the obtained force spectrum; non-linear alternatives to the Bell–Evans model should be used only if the forces measured do not scale linearly with the logarithm of the loading rate—as fitting a linear dependence with a non-linear function may result in fit parameters with little to no meaning.
5. Reagents Setup
- Assay buffer.
- UCP1–ATP example: 50 mM NaSO4, 10 mM MES, 10 mM Tris, 0.6 mM EDTA, adjusted to pH 7.2.
- SecYEG–SecA example: 50 mM Tris, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, adjusted to pH 7.0.
- Citric acid 1% (w/v). Dissolve 0.5 g of citric acid in 50 mL of Milli-Q H2O. Store at −20 °C in 2-mL aliquots for up to 2 years.
- Ethanolamine solution (1 M, pH 8.0). Dissolve 1.2 g of ethanolamine in 20 mL of Milli-Q H2O. Adjust the pH to 8.0 with 1 M HCl. Store at −25 °C in 0.5-mL aliquots for up to 2 years.
- Sodium cyanoborohydride (1 M solution with 20 mM NaOH). Working under a chemical hood, place 13 mg of NaCNBH3 into a polyethylene capped glass weighing bottle. Pipette 20 μL of 100 mM NaOH on top of the NaCNBH3 and mix well by pipetting. Then add 180 μL of Milli-Q water and mix by pipetting. If the balance used for weighing is outside of the chemical hood, first zero the balance using the empty weighing bottle with lid on, and then add ~13 mg of NaCNBH3 to the bottle under the hood. Cap the bottle and weigh. Adjust volumes of NaOH and Milli-Q water proportionately. CAUTION NaCNBH3 is highly toxic. Wear gloves and handle with care. Handle under a chemical hood during preparation. CAUTION Prepare fresh NaCNBH3 for each functionalization. Dispose of any remaining NaCNBH3 according to institutional regulations.
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