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Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy
采用浊点PEG玻璃表面法和全内反射显微镜进行固定化单分子成像   

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Abstract

This effective, robust protocol generates glass coverslips coated with biotin-functionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one day, and the coverslips can be stored for at least 1 month. We have confirmed that the PEG surfaces prepared according to the protocol are resistant to non-specific adsorption by a wide range of biomolecules (bacterial, mitochondrial, and human transcription factors, DNA, and RNA) and biological buffers.

Keywords: Single-molecule fluorescence(单分子荧光), Polyethylene glycol(聚乙二醇), Aminopropyltriethoxysilane(氨丙基三乙氧基硅烷), Cloud-point(云点), Surface chemistry(表面化学)

[Introduction] Single-molecule imaging methods of studying dynamics of biomolecules complement traditional ‘bulk’ biochemical methods by allowing real-time tracking of multi-step reactions without the need to synchronize the reagents (Weiss, 1999). In most single-molecule imaging methods, a biomolecule of interest is first labeled with a single fluorophore, the labeled biomolecule is then immobilized on an optically transparent surface (usually glass or silica), and detected as diffraction-limited image (‘spot’) using an optical microscope equipped with a high-sensitivity camera (Selvin and Ha, 2008). The surface immobilization serves two purposes. First, it permits tracking of molecular states on time scales longer than hundreds of milliseconds (otherwise, the biomolecule would diffuse out of the focal plane). Second, the surface permits excitation of fluorescence in total-internal-reflection geometry (Axelrod, 1981), which dramatically increases the signal-to-noise ratio of detection of molecules located in close proximity (<100 nm) to the surface (Selvin and Ha, 2008). Despite these clear advantages, surfaces are also the most common source of artifacts in single-molecule analysis (Visnapuu et al., 2008). For example, irreversible, non-specific adsorption of bio-molecules onto the imaging surface may reduce the effective concentration of the bio-molecule in the ‘bulk’, and thus perturb the rate of the biochemical reaction. Moreover, if the ‘sticky’ molecule is fluorescently labeled, the noise from numerous non-specifically ‘stuck’ fluorescent molecules may obscure the signal from specifically bound molecules, which will complicate data analysis. Finally, tethering a biomolecule to a surface greatly increases the effective concentration of that molecule with respect to the surface, and further increases the probability of non-specific binding due to repetitive ‘bumping’ of the tethered molecule during long-term tracking. Overall, the compatibility of a surface for the biomolecules of interest needs to be validated on a case by case basis, and there remains a great demand in the single-molecule imaging field for effective, robust methods of surface passivation.
The current protocol builds upon a technique first introduced by Ha and colleagues (Ha et al., 2002) which, in turn, built upon finding that polyethylene glycol (PEG) is most effective in creating anti-fouling surfaces (Prime and Whitesides, 1993), also see references in (Ostuni et al., 2001). In the original protocol by Ha et al. (2002), glass surfaces were first coated with a silanol-reactive aminopropyltriethoxysilane (APTES) to create amine groups, followed by deposition of a mixture of amine-reactive N-hydroxysuccinimide (NHS)-PEG (to create a passivation layer on the glass) and NHS-PEG-biotin (to create a handle for immobilization of bio-molecules for single-molecule tracking). In our protocol, PEG deposition is performed in cloud-point conditions, which reduces the size of the PEG globule and results in a denser, more adsorption-resistant, PEG layer (Kingshott et al., 2002). In addition, our protocol maximizes the reactivity of NHS-PEG during deposition. Furthermore, the protocol includes an end-capping step intended to eliminate residual amine groups remaining after PEG coupling, which we found to reduce non-specific adsorption of nucleic acids to surfaces in low-ionic-strength buffers required by some enzymes (Zhang et al., 2014). Finally, the protocol provides simple quality-control tips to help trouble-shooting. Despite these key improvements, we found that some proteins are still prone to non-specific adsorption to ’cloud point’ PEG surfaces. For instance, we found that the general transcription factor TFIID, a key component of the human transcription machinery, absorbs to ‘cloud-point’ PEG surfaces, whereas other five components of the basal human transcription machinery (TFIIB, TFIIF, TFIIE, TFIIH and RNA polymerase II) do not (Revyakin et al., 2012). Thus, we recommend testing the ‘cloud-point’ PEG surfaces using your specific buffers, biomolecules of interest, and biochemical activity assays.

Materials and Reagents

  1. Corning borosilicate cover glasses (24 x 40 mm, #1.5) (VWR International, catalog number: 48393-230 )
  2. PYREX crystallization dish (Thermo Fisher Scientific, catalog number: 08-741D )
  3. Nalgene 125 ml polypropylene jars (Thermo Fisher Scientific, catalog number: 11-815-10C )
  4. Two non-coring Syringe needles, luer, No 18, 6” long (Sigma-Aldrich, catalog number: Z102717-1EA )
  5. Parafilm (100 mm wide) (various suppliers)
  6. pH paper strips (0-14 range) (various suppliers)
  7. Double sided tape (3M formulation 4095) (McMaster, catalog number: 76665A67 )
  8. 30% hydrogen peroxide (Thermo Fisher Scientific, catalog number: H325-4 )
  9. 95-98% sulfuric acid (Sigma-Aldrich, catalog number: 320501-2.5 L )
  10. 99% 3-aminopropyltriethoxysilane (APTES) (Acros, catalog number: 430941000 ) ,
    Note: Stored under nitrogen in a container equipped with a rubber septum.
  11. Acetone (Chromasolv for HPLC) (Sigma-Aldrich, catalog number: 270725-1 L )
  12. Biotin-PEG-succinimidyl valerate [(bio-PEG-SVA) Mw = 5,000], (Laysan Bio)
    Note: Aliquoted into individual microtubes (1-2 mg/tube) by the end users and stored under desiccation at -80 °C.
  13. Methoxy-PEG-succinimidyl valerate [(mPEG-SVA), MW= 5,000] (Laysan Bio)
    Note: Aliquoted into individual microtubes (~5 mg/ tube) by the end users and stored under desiccation at -80 °C, with the mass of reagent written for each aliquot with 0.1 mg accuracy (e.g. 4.9 mg, 5.1 mg, and so on).
  14. Fluorescein-PEG-NHS (Nektar, catalog number: 1K4M0F02)
  15. Sulfosuccinimidyl acetate (Thermo Fisher Scientific, PierceTM, catalog number: 26777 )
  16. TRIONE ninhydrin reagent (Pickering Laboratories, catalog number: T100 )
  17. Potassium hydroxide (KOH) (semiconductor grade) (Sigma-Aldrich, catalog number: 306568-100 G )
  18. Sodium bicarbonate (NaHCO3) (ACS grade) (Sigma-Aldrich, catalog number: S6014-25 G )
  19. Potassium sulfate (K2SO4) (ACS grade) (Sigma-Aldrich, catalog number: 221325-500 G )
  20. Protein samples labeled at > 70% with a fluorophore suitable for single-molecule imaging with 532 nm or 640 nm excitation (e.g. Cy3, Alexa555, Atto633 and Alexa647N)
    Note: These should be the protein of interest to the end user. Commercially available ones can also be used.
  21. 0.5 M KOH (see Recipes)
  22. 0.5 M K2SO4 (see Recipes)
  23. 1 M NaHCO3 (see Recipes)
  24. Phosphate-buffered saline containing 0.1% Tween-20 (see Recipes)

Equipment

  1. Ceramic rack (for 12 coverslips) (Thomas Scientific, catalog number: 8542E40 )
  2. Forceps (for handling of ceramic staining racks) (Thermo Fisher Scientific, catalog number: 10-316C )
  3. Flat-tip tweezers (for handling of individual cover glasses) (Electron Microscopy Sciences, catalog number: 78335-35A )
  4. 250 ml PYREX beaker (various suppliers)
  5. A compressed nitrogen cylinder with a regulator and 0.2 micro filter (various suppliers)
  6. Two-Way Valve (PTFE) (Sigma-Aldrich, catalog number: 20926 )
  7. UV-Vis spectrophotometer (various suppliers)
  8. Horizontal platform shaker (common lab equipment) (various suppliers)
  9. Bath sonicator (Sonicsonline, Branson, catalog number: 2510 )
  10. Ultra pure water system (Merck Millipore Corporation)
  11. Diamond scriber (Ted Pella, catalog number: 54463 )
  12. Objective-type total-internal-reflection (TIR) microscope (Olympus TIRF objective lens NA = 1.49) equipped with an electron-multiplication CCD camera (Andor iXon+) and 532 and 640 nm laser excitation sources (various suppliers)

Procedure

  1. Piranha preparation and treatment to clean cover glasses
    Note: Be very careful as the Piranha solution used in this step is extremely corrosive. Follow your institution’s safety regulations. We recommend, at least, wearing a lab coat, a full-length rubber apron coat, long-sleeve butyl gloves, a full-face splash shield, and working under a chemical fume hood free of flammable organic chemicals. Use PYREX glassware for preparation of Piranha solutions (regular soda-lime glass beakers may crack upon heating when Pirahna solutions are prepared).
    1. Place 12 coverslips into ceramic rack using flat-tip tweezers.
    2. In a designated chemical fume hood, place two clean 250 ml PYREX beakers (beaker 1 and 2) in two separate crystallization dishes. The dishes serve as secondary containers to ensure safety.
    3. Very carefully add 1 part (50 ml) of 30% hydrogen peroxide (H2O2) to beaker 1, followed by 3 parts (150 ml) of concentrated sulfuric acid (H2SO4). Gently stir to mix the solution with a clean glass rod. The solution will immediately form bubbles and heat up to ~100 °C.
    4. Using forceps, very carefully transfer the rack with the coverslips into beaker 1 and incubate for 30 min. To ensure safety, leave a note indicating that Piranha is in use.
    5. Repeat Piranha treatment one more time with a fresh Piranha solution in beaker 2.
    6. Transfer the coverslips from beaker 2 into a Nalgene 125 ml polypropylene jar filled with double-distilled water. Dispose of piranha waste according to your institution’s regulations. Rinse the coverslips copiously with double-distilled water until pH stabilizes (verified by pH paper). The cleaned coverslips can be stored in double-distilled water without noticeable changes in reactivity and fluorescent background for at least 1 month.
    7. Quality control.
      1. Surface hydrophilicity. Piranha-treated coverslips become uniformly hydrophilic, which can be qualitatively verified by dipping a coverslip in water using flat-end tweezers, taking it out vertically, and observing water slowly receding as a uniform sheet, and forming Young’s rings before drying out. In contrast, untreated coverslips form patches of water when dipped into and taken out of water
      2. Fluorescent background. After Piranha treatment, blow-dry a coverslip with pressurized pure nitrogen (see step C12) and place it onto a total-internal-reflection microscope. One should observe essentially no fluorescent spots (<3 identifiable ‘spots’ per 100 x 100 micron area) under typical single-molecule imaging conditions (excitation with 532 and 640 nm sources at density ~0.5 kW/cm2, imaging in 580/60 nm and 670/45 nm optical bands, sampling rate 2.5 Hz).

  2. Mild KOH etching to maximize the density of silanol groups on the glass surface (Iler, 1979)
    1. Transfer the Piranha-treated coverslips from water to a clean 250 ml polypropylene jar containing 125 ml of 0.5 M KOH, then sonicate for 1 h in a bath sonicator.
      1. Be sure that the water level is up to the marked operating level, and is about level with the solution inside the jar.
      2. Ensure that the temperature does not exceed 40 °C by supplying ice (100~200 g by estimation) at t = 0 and t = 30 min during sonication. Otherwise the bath will heat up, which accelerates etching of the coverslips and creates micron-size pits in the glass surfaces.
      3. Transfer the coverslips back into the polypropylene jar containing water and copiously rinse the coverslips with double-distilled water until pH stabilizes, as verified by pH paper.

  3. Silanization to create amine groups on glass surface
    Note: In this step, the glass surface is functionalized with amine groups by incubating in a solution of APTES. Although APTES is one the best studied silanization agents, there is currently no consensus on the best method to cost-effectively create molecularly homogenous, reactive, stable APTES films. The parameter space for APTES deposition conditions is vast, as the quality of films has been shown to depend on the substrate (e.g. glass, silica, or oxidized silicon), surface cleaning method, deposition phase (e.g. liquid or vapor), water content of the solvent and the surface, concentration of APTES, temperature, incubation times, and annealing conditions [for references, see Kim et al. (2009)]. Our procedure is based on the simple technique introduced to the single-molecule imaging field by Ha and colleagues (Ha et al., 2002). In our hands, more sophisticated deposition methods did not create APTES surfaces superior to the ones prepared per Ha et al. (2002).

    1. Under a fume hood, prepare three clean 125 ml polypropylene jars (maximal capacity of ~170 ml) containing 125 ml acetone.
    2. Connect a non-coring needle to a 10 cc gas-tight glass syringe via a PTFE valve. Close the PTFE valve. Insert the needle through the septum of the APTES bottle and ensure that the needle orifice is below the surface of the APTES. Create slight positive pressure inside the APTES sealed bottle using a nitrogen line and a second non-coring needle. Open the PTFE valve. Withdraw 3.75 ml of APTES with the syringe and close the PTFE valve. Remove the needle from the bottle, open the PTFE valve, and add the APTES into the 125 ml of acetone in one of the jars. Gently stir to prepare 3% APTES.
    3. Using forceps, remove the rack with coverslips from double-distilled water, and dip the rack successively into the two 125 ml polypropylene jars containing acetone, spending ~10 sec in each jar with gentle agitation. Then transfer immediately into the freshly prepared 3% APTES solution. Do not allow acetone to dry between transfers. Place the APTES container on a horizontal platform shaker and incubate for 1 h.
      Note: Immediately after use, during silanization, rinse the syringe, the syringe needles, and the PTFE valve sequentially with acetone and double-distilled water, and then blow with dry nitrogen. Also, during silanization, prepare PEG solutions (steps D14-15).
    4. After silanization, wash the coverslips with acetone by dipping the rack in the two jars containing acetone (see step C11) in reverse order and place the rack back into double-distilled water. Copiously rinse with double-distilled water. Do not let the coverslips spend more than 5 min in water. Blow the coverslips dry one-by-one with nitrogen. To that end, hold a coverslip on its corner with the flat-end tweezers, and direct the nitrogen gas flow across the surface of the coverslip towards the corner held by the tweezers. When dry, place the coverslip on a piece of Parafilm positioned on a flat, clean surface. Mark the upper surface of the coverslip by scoring the upper-left corner with a diamond scriber. The coverslips are now ready for PEGylation.
      Notes:
      1. We usually carry out PEGylation in a clean environment (e.g. a positive-pressure HEPA-filtered room), but we also have had success in a regular environment if PEGylation was carried out immediately after treatment by APTES. We typically do not store amine-treated coverslips.
      2. Many APTES deposition protocols include a high-temperature curing step following silanization, meant to form stable covalent bonds between physisorbed APTES molecules (Plueddemann, 1982). However, on the molecular scale, curing may lead to lateral rearrangement of APTES molecules and create islands of unmodified glass (Kim et al., 2009). In our hands, curing offered no additional improvement in surface quality in terms of preventing non-specific adsorption of biomolecules.
    5. Quality control: Concentration of amine groups. You can measure the concentration of amine groups on coverslip surfaces using TRIONE ninhydrin reagent. To that end, crush one coverslip by placing it into a 50 ml Falcon tube and centrifuging at 2,000 x g in a swing-bucket centrifuge for 1 min. Then fill the tube with 1 ml of the ninhydrin reagent and follow the manufacturer’s protocol to spectrophotometrically quantify the average density of amine groups on the coverslip surface (for 24 x 40 mm coverslips, with the thickness of ~0.15 mm, the total surface area is 1,940 mm2). Using standard dilutions of APTES as create a calibration curve, we typically get 3 amine groups per nm2.

  4. Treatment of amine-glass surfaces with NHS-PEG at cloud-point conditions
    Notes:
    1. The procedure builds upon the method introduced by Ha et al. (2002), with modifications to maximize the density of PEG molecules, and to block and neutralize positively charged amine groups after PEG deposition (section E). Thus, amine-treated coverslips are coupled to succinimidyl-PEG at pH 9.0 in a bicarbonate buffer containing 0.45 M K2SO4, at 10.0 % NHS-PEG (w/v). The high salt and the high PEG concentration bring the PEG solution just below its ‘cloud point’, which maximizes the density of the PEG layer on the surface (Kingshott et al., 2002).
    2. NHS group hydrolyzes in aqueous buffers, which makes NHS-PEG non-reactive. The lifetime of the NHS group is between a few seconds and a few minutes, depending on the nature of the NHS-PEG linker, pH, and temperature (Hermanson, 2008). For example, we found that NHS-SVA hydrolyses with a half-life of ~5 min at pH 9.0 at room temperature, which is a good compromise between the reactivity of the NHS group and the reactive species lifetime. Thus, our protocol minimizes the time the reactive NHS-SVA species spends at pH=9.0 prior to addition to the amine-glass surface. Specifically, we initially dissolve dry PEG-SVA at pH 6.0, at which the NHS group remains unhydrolyzed for at least 1 h. Then, immediately before addition of the PEG solution to the glass surface, we bring the pH up to 9.0. If you are using a non-SVA linker, you have to optimize the pH of the reaction to achieve the NHS lifetime of ~5 min.
    3. The lifetime of the reactive NHS group can be measured spectrophotometrically by monitoring the kinetics of accumulation of free NHS upon hydrolysis [free NHS strongly adsorbs at 260 nm (Miron, 1982)]. We also highly recommend measuring the percentage of reactive PEG-NHS in new batches of purchased PEG reagents. We have had cases in which completely hydrolyzed, non-reactive PEGs had been shipped by major suppliers.
    1. This step is best done during the APTES incubation at step C11. Take out 6 single-use aliquots of dry mPEG-SVA from storage at -80 °C. Each aliquot should be about 5 mg, which is sufficient to treat two coverslips. The mass of dry mPEG-SVA in the 6 aliquots should have been pre-written on each tube with 0.1 mg accuracy prior to storage at -80 °C (e.g. 4.9, 5.0, 5,1, 4.9, 5.0, and 5.1 mg for 6 tubes). In addition, take out 1-2 mg of biotin-PEG-SVA from storage. Let all tubes warm up to room temperature.
    2. This step is best done during the APTES incubation at step C11. Calculate the volume of 0.5 M K2SO4 solution to add to each mPEG-SVA tube by multiplying the aliquot mass by 8 (e.g. the tube containing 4.9 mg will require 4.9 x 8 = 39.2 μl of K2SO4 solution) and record all volumes in a notebook. Sum up all the volumes, to get the minimal volume of 0.5 M K2SO4 required to make six PEG solutions [e.g., for 6 tubes containing 4.9, 5.0, 5,1, 4.9, 5.0, and 5.1 mg mPEG-SVA one needs (4.9 + 5.0 + 5.1 + 4.9 + 5.0 + 5.1) x 8 = 240 μl of K2SO4]. Based on the calculated minimal volume, prepare sufficient amount of 0.5% biotin-PEG-SVA solution in 0.5 M K2SO4 (e.g. dissolve 1.3 mg of biotin-PEG-SVA with 260 μl 0.5 M K2SO4). Pipet the pre-calculated volumes of the 0.5% biotin-PEG-SVA solution into each of the 6 mPEG-SVA aliquots (e. g. add 39.2 μl to the 4.9 mg tube, and so on), vortex for 10 sec, and briefly spin with a tabletop centrifuge. Each tube now should have a clear solution and a small (~5 μl in volume) pellet of PEG on the bottom.
      Note: PEG precipitation is expected, because the PEG solution is currently at 11.6% concentration (w/v), which is above its ‘cloud point’ (10% w/v).
      The tubes can remain at room temperature for at least 1 h without appreciable hydrolysis of the NHS group while the coverslips are being treated with APTES and laid out for PEGylation.
    3. With all APTES-treated coverslips laid out for PEGylation, set up the PEGylation reactions using one PEG-NHS aliquot at a time. To that end, add 1/8 of 1 M NaHCO3 (pH 9.0) to the PEG solution in K2SO4 (e.g. add 4.9 μl of NaHCO3 to a tube that originally contained a 4.9 mg mPEG-SVA aliquot, and now contains 39.2 μl of the biotin-PEG-SVA solution), quickly mix by pipetting up and down, and deposit the whole solution in the center of a dry APTES-treated coverslip. Cover the drop with another coverslip carefully, making sure that the scored surfaces of both coverslips face towards the PEG solution, and avoiding formation of bubbles (by practicing). Repeat for the rest five pairs of coverslip and incubate for 30 min at room temperature.
      Note: The PEG solution appears clear after addition of NaHCO3, because the total PEG concentration is now just below the ‘cloud point’. We noticed that additional coating of coverslips with a fresh solution of PEG does not improve the quality of surfaces (in terms of non-specific adsorption of biomolecules).
    4. Carefully separate the coverslip pairs using tweezers and place individual coverslips back into the ceramic rack in the double-distilled water. Rinse extensively with water until there is no more foaming of the solution.
      Quality control: Density of PEG molecules. You can estimate the packing density of the PEG molecules on the glass surface by depositing fluorescein-PEG-NHS in the same conditions as described above, and measuring the absorbance at 494 nm using a high-sensitivity double-beam spectrophotometer (e.g. Perkin Elmer Lambda 35). The molecules density = [(OD494/extinction coefficient) * 6.02 x 1023 mol−1]/area. By this estimation, the average packing density should be consistent with the radius of gyration of a Mw=5,000 PEG molecule at cloud point [R=2.8 nm (Dalsin et al., 2005)].

  5. Blocking of unreacted amines with acetyl groups
    Note: Due to the bulkiness of PEG molecules, only a fraction of amine groups on the surface reacts with NHS-PEG. As a result, the remaining amines contribute to non-specific adsorption of negatively charged molecules, such as DNA, which is problematic in buffers of low ionic strengths. Thus, we cap the unreacted amines with NHS-acetate to convert them to neutral, stable amides.
    1. Blow-dry 12 PEGylated coverslips as described in step C12. Place dry coverslips on Parafilm, with the PEGylated surface facing up.
    2. Immediately prior to use, dissolve 3 mg of sulfo-NHS-acetate in 300 μl of 0.1 M NaHCO3 (pH 9.0), deposit 50 μl drops of the solution onto the 6 PEG coverslips, and cover with the remaining 6 coverslips, with the PEGylated surface facing the solution. Incubate for 30 min at room temperature. Rinse coverslips with double-distilled water, blow-dry, and store dry at -80 °C.

  6. Testing fluorescent background of PEGylated surfaces and non-specific absorption of biomolecules
    Note: The following protocol is for a rapid test of non-specific ‘stickiness’ of the surface to fluorescently labeled biomolecules. However, this test does not address the biological activity of surface-immobilized molecules (for instance, the activity of a transcription factor on a surface-immobilized DNA). Thus, the most relevant test of surface quality is a biochemical assay of the activity of surface-bound biomolecule, performed side-by-side with a positive control in which the activity of the same amount of biomolecules is measured in solution-based conditions (Revyakin et al., 2012).
    1. Create 5 open wells on a coverslip using 1 mm-wide strips of double-sided tape (by simply sticking 6 strips on to the modified side), and place the coverslip on an objective-type TIRF microscope. The 5 wells allow testing of 5 different biomolecules.
    2. Deposit a 5 μl drop of PBST into a well, turn on the excitation laser, and focus the microscope onto the surface. Acquire a movie to measure the fluorescent background on the surface, under single-molecule imaging conditions (e.g. excitation with 532 and 640 nm sources at density ~0.5 kW/cm2, imaging in 580/60 nm and 670/45 nm optical bands, sampling rate 2.5 Hz). In our hands, silanization and PEGylation increase the number of fluorescent background spots ~3 fold in comparison to Piranha-treated glass (i.e. an increase from ~3 spots to ~10 spots per 100 x 100 μm field of view), which is an acceptable level of background for most single-molecule imaging experiments. Allow the background spots to photobleach.
    3. Prepare a 10 nM solution of the fluorescently labeled test protein in PBST, and deposit 20 μl of the solution on top of the 5 μl PBST drop already in the well. Acquire a movie to quantify the non-specific adsorption of the molecule to the surface. For simplicity, in this test we do not supply oxygen scavenger to the solution of test protein. Thus, to avoid immediate photobleaching upon sticking of a molecule to the surface (which will lead to over-estimation of surface quality) use minimal laser power (30~100 W/cm2) and fluorescent labels that photobleach in >5 seconds in the absence of oxygen scavengers (e.g. Cy3 and Atto633). For a good-quality surface, we typically observe a ‘cloud’ of 10 nM biomolecules rapidly diffusing in the bulk solution (at 2.5 Hz acquisition rate), and ~10 single-molecule limited spots in any given movie frame (100 x 100 μm field of view). No additional accumulation of spots should be observed within 10 min (with the excitation light switched off, and then turned back on again), indicating that non-specific adsorption is rare and reversible. Repeat the test with other biomolecules of interest using the remaining 4 wells.

Recipes

  1. 0.5 M KOH (per liter)
    28 g KOH
    Note: Solution can be re-used.
  2. 0.5 M K2SO4 (pH 6.0) (per liter)
    174 g K2SO4
    Note: Store at room temperature. If your double-distilled water supply is acidic, no pH adjustment of the K2SO4 solution is necessary. If your double-distilled water supply is basic, adjust pH to 6.0 using diluted H2SO4.
  3. 1 M NaHCO3 (pH 9.0) (10x) (per liter)
    82 g NaHCO3
    Note: Adjust pH to 9.0 with NaOH and stored in single-use aliquots at -80 °C.
  4. Phosphate-buffered saline containing 0.1% Tween (PBST) (per liter)
    2.0 g KCl
    2.4 g KH2PO4
    80 g NaCl
    14.4 g Na2HPO4
    1 ml Tween 20

Acknowledgments

We thank the groups of Steven Chu and Robert Tjian for support (University of California, Berkeley, and Janelia Farm Research Camus, supported by the NIH grant 1P01CA112181-01A1 and Howard Hughes Medical Institute).

References

  1. Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89(1): 141-145.
  2. Dalsin, J. L., Lin, L., Tosatti, S., Voros, J., Textor, M. and Messersmith, P. B. (2005). Protein resistance of titanium oxide surfaces modified by biologically inspired mPEG-DOPA. Langmuir 21(2): 640-646.
  3. Ha, T., Rasnik, I., Cheng, W., Babcock, H. P., Gauss, G. H., Lohman, T. M. and Chu, S. (2002). Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419(6907): 638-641.
  4. Hermanson, G. T. (2008). Bioconjugation techniques. Academic Press, 10, p.0123705010.
  5. Iler, R. K. (1979). The chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry. Wiley 896.
  6. Kim, J., Seidler, P., Wan, L. S. and Fill, C. (2009). Formation, structure, and reactivity of amino-terminated organic films on silicon substrates. J Colloid Interface Sci 329(1): 114-119.
  7. Kingshott, P., Thissen, H. and Griesser, H. J. (2002). Effects of cloud-point grafting, chain length, and density of PEG layers on competitive adsorption of ocular proteins. Biomaterials 23(9): 2043-2056.
  8. Miron, T. and Wilchek, M. (1982). A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters. Anal Biochem 126(2): 433-435.
  9. Ostuni, E., Chapman, R. G., Holmlin, R. E., Takayama, S. and Whitesides, G. M. (2001). A survey of structure - property relationships of surfaces that resist the adsorption of protein. ASC Publications 17(9): pp.5605-5620.
  10. Plueddemann, E. (1982). Silane coupling agents, Springer Netherlands.
  11. Prime, K. L. and Whitesides, G. M. (1993). Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using self-assembled monolayers. J Am Chem Soc 115(23): 10714-10721.
  12. Revyakin, A., Zhang, Z., Coleman, R. A., Li, Y., Inouye, C., Lucas, J. K., Park, S. R., Chu, S. and Tjian, R. (2012). Transcription initiation by human RNA polymerase II visualized at single-molecule resolution. Genes Dev 26(15): 1691-1702.
  13. Selvin, P. R. and Ha, T. (2008). Single-molecule techniques : a laboratory manual. Cold Spring Harbor Laboratory Press.
  14. Visnapuu, M. L., Duzdevich, D. and Greene, E. C. (2008). The importance of surfaces in single-molecule bioscience. Mol Biosyst 4(5): 394-403.
  15. Weiss, S. (1999). Fluorescence spectroscopy of single biomolecules. Science 283(5408): 1676-1683.
  16. Zhang, Z., Revyakin, A., Grimm, J. B., Lavis, L. D. and Tjian, R. (2014). Single-molecule tracking of the transcription cycle by sub-second RNA detection. Elife 3: e01775.

简介

这种有效的,稳健的方案产生涂覆有生物素官能化聚乙二醇(PEG)的玻璃盖玻片,使得玻璃表面对生物分子的非特异性吸收具有抗性,并允许固定生物分子以用于随后的单分子跟踪生化反应。 协议可以在一天内完成,盖玻片可以存储至少1个月。 我们已经证实,根据方案制备的PEG表面可以通过广泛的生物分子(细菌,线粒体和人转录因子,DNA和RNA)和生物缓冲液的非特异性吸附抗性。
【背景】研究生物分子动力学的单分子成像方法通过允许实时跟踪多步反应而不需要同步试剂来补充传统的“大量”生物化学方法(Weiss,1999)。在大多数单分子成像方法中,首先用单个荧光团标记感兴趣的生物分子,然后将标记的生物分子固定在光学透明的表面(通常为玻璃或二氧化硅)上,并且被检测为衍射受限图像(“斑点”)使用配有高灵敏度相机的光学显微镜(Selvin和Ha,2008)。表面固定有两个目的。首先,它允许在超过几百毫秒的时间尺度上跟踪分子态(否则,生物分子将扩散到焦平面外)。第二,表面允许在全内反射几何中激发荧光(Axelrod,1981),其显着地增加了与表面接近(<100nm)位置的分子的检测的信噪比(Selvin和哈,2008)。尽管有这些明显的优势,表面也是单分子分析中最常见的人为因素(Visnapuu et al。,2008)。例如,生物分子在成像表面上的不可逆的非特异性吸附可能会降低生物分子在“体积”中的有效浓度,从而扰乱生化反应的速率。此外,如果“粘性”分子被荧光标记,则来自许多非特异性“卡住”荧光分子的噪声可能会遮蔽来自特异结合分子的信号,这将使数据分析复杂化。最后,将生物分子束缚到表面大大增加了该分子相对于表面的有效浓度,并且由于在长期追踪期间由于重复的拴系分子的“碰撞”而进一步增加非特异性结合的可能性。总体而言,感兴趣的生物分子的表面的相容性需要根据具体情况进行验证,并且单分子成像领域对于表面钝化的有效,鲁棒的方法仍然存在很大的需求。
目前的方案建立在Ha及其同事首先引入的技术(Ha et al。,2002)的基础上,后者又发现聚乙二醇(PEG)在产生防污面方面最有效(Prime和Whitesides,1993 ),也参见(Ostuni等人,2001)中的参考文献。在Ha等人的原始协议中(2002)中,首先用硅烷醇反应性氨基丙基三乙氧基硅烷(APTES)涂覆玻璃表面以产生胺基,随后沉积胺反应性N-羟基琥珀酰亚胺(NHS)-PEG的混合物(以在玻璃上产生钝化层)和NHS-PEG-生物素(以产生用于固定生物分子用于单分子跟踪的手柄)。在我们的方案中,PEG沉淀在浊点条件下进行,这降低了PEG球的尺寸,并导致更致密,更耐吸附的PEG层(Kingshott等,2002)。此外,我们的协议最大限度地提高了NHS-PEG在沉积过程中的反应性。此外,该方案包括旨在消除PEG偶联之后残留的残留胺基的封端步骤,我们发现这些步骤减少了一些酶所需的低离子强度缓冲液中核酸对表面的非特异性吸附(Zhang et al ,2014)。最后,该协议提供简单的质量控制提示来帮助故障排除。尽管有这些关键的改进,我们发现一些蛋白质仍然易于非特异性吸附到“浊点”PEG表面。例如,我们发现一般转录因子TFIID(人转录机制的关键组分)吸收了“云点”PEG表面,而基础人转录机制的其他五个成分(TFIIB,TFIIF,TFIIE,TFIIH和RNA聚合酶II)(Revyakin等,2012)。因此,我们建议您使用您的特定缓冲液,感兴趣的生物分子和生物化学活性测定法来测试“云点”PEG表面。

关键字:单分子荧光, 聚乙二醇, 氨丙基三乙氧基硅烷, 云点, 表面化学

[简介] 研究生物分子动力学的单分子成像方法通过允许实时跟踪多步反应而不需要同步试剂来补充传统的"大量"生化方法(Weiss,1999) 。在大多数单分子成像方法中,首先用单个荧光团标记感兴趣的生物分子,然后将标记的生物分子固定在光学透明表面(通常是玻璃或二氧化硅)上,并作为衍射限制图像("点")检测使用装备有高灵敏度相机的光学显微镜(Selvin和Ha,2008)。表面固定有两个目的。首先,其允许在长于数百毫秒的时标上跟踪分子状态(否则,生物分子将扩散出焦平面)。第二,表面允许在全内反射几何中激发荧光(Axelrod,1981),其显着增加了与表面非常接近(<100nm)的分子的检测的信噪比(Selvin和Ha,2008)。尽管有这些明显的优点,但表面也是单分子分析中最常见的人工产物来源(Visnapuu等人,2008)。例如,生物分子不可逆地非特异性吸附到成像表面上可能降低生物分子在"本体"中的有效浓度,并因此干扰生化反应的速率。此外,如果"粘性"分子被荧光标记,来自许多非特异性"固定"荧光分子的噪声可能遮蔽来自特异性结合分子的信号,这将使数据分析复杂化。最后,将生物分子束缚在表面上大大增加了该分子相对于表面的有效浓度,并且进一步增加了由于在长期跟踪期间重复"碰撞"拴系分子而引起的非特异性结合的可能性。总的来说,需要根据情况来验证表面对于感兴趣的生物分子的相容性,并且在单分子成像领域中对于表面钝化的有效,鲁棒的方法仍然有很大的需求。
  目前的方案建立在Ha和同事首先介绍的技术(Ha等人,2002)上,其又基于发现聚乙二醇(PEG)在产生防污垢方面最有效表面(Prime和Whitesides,1993),也参见参考文献(Ostuni等人,2001)。在Ha等人(2002)的原始方案中,玻璃表面首先用硅烷醇反应性氨基丙基三乙氧基硅烷(APTES)涂覆以产生胺基,随后沉积胺反应性N - 羟基琥珀酰亚胺(NHS)-PEG(以在玻璃上产生钝化层)和NHS-PEG-生物素(以产生用于固定化用于单分子跟踪的生物分子的手柄)。在我们的方案中,在浊点条件下进行PEG沉积,这降低了PEG球的尺寸并导致更致密,更耐吸附的PEG层(Kingshott等人,2002) 。此外,我们的协议最大化NHS-PEG沉积期间的反应性。此外,该方案包括旨在消除在PEG偶联后剩余的残基胺基团的封端步骤,我们发现其减少了一些酶所需的低离子强度缓冲液中核酸对表面的非特异性吸附(Zhang et al。,2014)。最后,协议提供简单的质量控制提示,以帮助排除故障。尽管这些关键的改进,我们发现一些蛋白质仍然倾向于非特异性吸附到"浊点"PEG表面。例如,我们发现一般转录因子TFIID,人类转录机制的关键组成部分,吸收到"浊点"PEG表面,而基础人类转录机制的其他五个组件(TFIIB,TFIIF,TFIIE,TFIIH和RNA聚合酶II)没有(Revyakin等人,2012)。因此,我们建议使用您的特定缓冲液,感兴趣的生物分子和生化活性测定测试'浊点'PEG表面。

材料和试剂

  1. Corning硼硅酸盐盖玻璃(24×40mm,#1.5)(VWR International,目录号:48393-230)
  2. PYREX结晶皿(Thermo Fisher Scientific,目录号:08-741D)
  3. Nalgene 125ml聚丙烯罐(Thermo Fisher Scientific,目录号:11-815-10C)
  4. 两个非取芯注射器针,Luer,No 18,6"长(Sigma-Aldrich,目录号:Z102717-1EA)
  5. 石蜡膜(100mm宽)(各供应商)
  6. pH纸条(0-14系列)(各供应商)
  7. 双面胶带(3M制剂4095)(McMaster,目录号:76665A67)
  8. 30%过氧化氢(Thermo Fisher Scientific,目录号:H325-4)
  9. 95-98%硫酸(Sigma-Aldrich,目录号:320501-2.5L)
  10. 99%3-氨基丙基三乙氧基硅烷(APTES)(Acros,目录号:430941000),
    注意:在氮气下储存在装有橡胶隔膜的容器中。
  11. 丙酮(Chromasolv for HPLC)(Sigma-Aldrich,目录号:270725-1L)
  12. 生物素-PEG-琥珀酰亚胺戊酸酯[(生物-PEG-SVA)M sub = 5000],(Laysan Bio)
    注意:由最终用户分装到单独的微管(1-2mg /管)中并在-80℃下干燥储存。
  13. 甲氧基-PEG-琥珀酰亚胺基戊酸酯[(mPEG-SVA),M sub W = 5000](Laysan Bio)
    注意:由终端用户分装到单独的微管(?5mg /管)中并在-80℃下干燥储存,每个等分试样的试剂质量为0.1mg精确度(例如4.9mg,5.1mg ,等等)。
  14. 荧光素-PEG-NHS(Nektar,目录号:1K4M0F02)
  15. 磺基琥珀酰亚胺基乙酸酯(Thermo Fisher Scientific,Pierce TM,目录号:26777)
  16. TRIONE茚三酮试剂(Pickering Laboratories,目录号:T100)
  17. 氢氧化钾(KOH)(半导体级)(Sigma-Aldrich,目录号:306568-100G)
  18. 碳酸氢钠(NaHCO 3)(ACS级)(Sigma-Aldrich,目录号:S6014-25G)
  19. 硫酸钾(K 2 SO 4)(ACS级)(Sigma-Aldrich,目录号:221325-500G)
  20. 标记为> 70%,具有适用于具有532nm或640nm激发的单分子成像(例如Cy3,Alexa555,Atto633和Alexa647N)的荧光团。
    注意:这些应该是最终用户感兴趣的蛋白质。也可以使用市售的。
  21. 0.5 M KOH(见配方)
  22. (参见配方)。
  23. 1 M NaHCO 3 3(参见配方)
  24. 含有0.1%Tween-20的磷酸盐缓冲盐水(见配方)

设备

  1. 陶瓷架(12盖玻片)(Thomas Scientific,目录号:8542E40)
  2. 镊子(用于处理陶瓷染色架)(Thermo Fisher Scientific,目录号:10-316C)
  3. 平头镊子(用于处理个别盖玻片)(电子显微镜科学,目录号:78335-35A)
  4. 250 ml PYREX烧杯(各供应商)
  5. 带有调节器和0.2微过滤器的压缩氮气瓶(各种供应商)
  6. 二通阀(PTFE)(Sigma-Aldrich,目录号:20926)
  7. 紫外可见分光光度计(各供应商)
  8. 水平振动台(普通实验室设备)(各供应商)
  9. Bath超声波仪(Sonicsonline,Branson,目录号:2510)
  10. 超纯水系统(Merck Millipore Corporation)
  11. 钻石雕刻机(Ted Pella,目录号:54463)
  12. 配备有电子倍增CCD照相机(Andor iXon +)和532和640nm激光激发源(各种供应商)的物镜型全内反射(TIR)显微镜(奥林巴斯TIRF物镜NA = 1.49)

程序

  1. Piranha清洁护目镜的准备和处理
    注意:要非常小心,因为本步骤中使用的Piranha溶液具有极强的腐蚀性。遵循机构的安全规定。我们建议至少佩戴实验室外套,全长橡胶围裙,长袖丁基手套,全脸防溅罩,并在化学通风橱下工作,不含易燃有机化学品。使用PYREX玻璃器皿制备Piranha溶液(常规的钠钙玻璃烧杯在制备Pirahna溶液时可能会发热)。
    1. 使用平头镊子将12盖玻片放入陶瓷架。
    2. 在指定的化学通风橱中,放置两个干净的250毫升PYREX烧杯 ?(烧杯1和2)在两个单独的结晶皿中。盘子 作为次级容器,以确保安全
    3. 很小心 向烧杯1中加入1份(50ml)30%过氧化氢(H 2 O 2),随后 ?加入3份(150ml)浓硫酸(H 2 SO 4)。轻轻搅拌 以将溶液与干净的玻璃棒混合。解决办法 立即形成气泡并加热至?100℃
    4. 使用镊子, 非常仔细地将机架与盖玻片转移到烧杯1和 孵育30分钟。为了确保安全,请留下注释 比拉鱼正在使用。
    5. 在烧杯2中用新鲜的Piranha溶液再次重复Piranha处理一次。
    6. 将盖玻片从烧杯2转移到Nalgene 125毫升 聚丙烯罐装满双蒸水。处理食人鱼 ?根据您所在机构的规定进行废物处理。冲洗盖玻片 ?大量用双蒸水直至pH稳定(通过验证 pH试纸)。清洁的盖玻片可以储存在双蒸 水,反应性和荧光没有显着变化 背景至少1个月。
    7. 质量控制。
      1. 表面 ?亲水性。食人鱼处理的盖玻片变得均匀 亲水的,其可以通过浸渍盖玻片定性验证 在水中使用平端镊子,垂直取出,和 观察水缓慢后退作为均匀的片材,并形成杨氏 环干燥前。相比之下,未处理的盖玻片形成贴片 的水浸入和取出水
      2. 荧光灯 背景。比拉鱼治疗后,吹干盖玻片 加压纯氮(参见步骤C12),并将其放置到a 全内反射显微镜。人们应该基本上观察不到 荧光斑点(每100×100微米面积<3个可识别的斑点) ?在典型的单分子成像条件下(用532激发) 和密度为?0.5kW/cm 2的640nm光源,在580/60nm的成像和 670/45nm光带,采样率2.5Hz)。

  2. 轻度KOH蚀刻以使玻璃表面上的硅烷醇基团的密度最大化(Iler,1979)
    1. 转移Piranha处理的盖玻片从水到一个干净的250毫升 聚丙烯罐中,含有125ml的0.5M KOH,然后超声处理1小时 在浴超声波仪。
      1. 确保水位达到标记的操作水平,并且与罐内的溶液接近水位。
      2. 通过供冰确保温度不超过40°C (在估计时为100?200g)在超声处理期间在t = 0和t = 30分钟。 否则,浴将加热,这加速蚀刻 盖玻片并在玻璃表面中产生微米级凹坑。
      3. 将盖玻片转移回含有水的聚丙烯罐中 ?并用双蒸水大量冲洗盖玻片直到pH ?稳定,如通过pH试纸证实的。

  3. 硅烷化在玻璃表面上产生胺基
    注意:在此步骤中,通过在APTES的溶液中温育,将玻璃表面用胺基官能化。尽管APTES是研究最好的硅烷化剂之一,但是目前还没有关于最佳方法的经济有效地产生分子均匀,反应性,稳定的APTES膜的共识。 APTES沉积条件的参数空间是巨大的,因为膜的质量已经显示取决于基底(例如玻璃,二氧化硅或氧化的硅),表面清洁方法,沉积阶段(例如液体或蒸气),水含量溶剂和表面,APTES的浓度,温度,孵育时间和退火条件[参考文献,参见Kim et al。 (2009)]。我们的程序是基于Ha和同事介绍的单分子成像领域的简单技术(Ha等人,2002)。在我们的手中,更复杂的沉积方法不产生优于根据Ha等人制备的APTES表面。 (2002)。

    1. 在通风橱下,准备三个干净的125毫升聚丙烯瓶(最大容量?170毫升)含有125毫升丙酮。
    2. 通过a将非取芯针连接到10 cc气密玻璃注射器 ?PTFE阀。关闭PTFE阀。将针头插入隔垫 的APTES瓶,并确保针孔在下面 表面的APTES。在APTES内部产生微小的正压 使用氮气管线和第二非取芯针密封瓶。打开 ?PTFE阀。用注射器取出3.75ml的APTES并关闭 PTFE阀。从瓶中取出针头,打开PTFE阀, 并将APTES加入其中一个瓶中的125ml丙酮中。轻轻地 搅拌制备3%APTES。
    3. 使用镊子,拆下机架 从双蒸水盖玻片,并连续浸渍机架 进入两个125毫升含有丙酮的聚丙烯罐中,花费?10 秒,并轻轻搅拌。然后立即转入 新鲜制备的3%APTES溶液。不要让丙酮晾干 之间转移。将APTES容器放在水平平台上 振摇并孵育1小时。
      注意:使用后,期间 硅烷化,冲洗注射器,注射器针头和PTFE阀 ?依次用丙酮和双蒸水洗涤,然后吹风 用干燥氮气。此外,在硅烷化期间,制备PEG溶液 (步骤D14-15)。
    4. 硅烷化后,用盖玻片洗涤 丙酮通过将架子浸入含有丙酮的两个瓶子中(参见步骤 ?C11),并将机架放回双馏份 水。大量用双蒸水冲洗。不要让 盖玻片在水中花费超过5分钟。吹干盖玻片 逐一与氮。为此,在它的角落上盖一个盖玻片 与平端镊子,并引导氮气流过 盖片的表面朝向由镊子保持的角部。什么时候 干燥,将盖玻片放在一片位于平面上的石蜡膜上, 清洁表面。通过划痕标记盖玻片的上表面 左上角有一个钻石雕刻机。盖玻片现在准备好了 用于聚乙二醇化。
      注意:
      1. 我们通常在a中进行PEG化 ?清洁环境(例如正压HEPA过滤的房间),但我们 ?也有在常规环境中的成功,如果聚乙二醇化 在APTES治疗后立即进行。我们通常不会 存储胺处理的盖玻片。
      2. 许多APTES沉积协议 包括在硅烷化之后的高温固化步骤 在物理吸附的APTES分子之间形成稳定的共价键 (Plueddemann,1982)。然而,在分子规模上,可能导致固化 ?APTES分子的侧向重排和创建岛 未改性玻璃(Kim等人2009)。在我们手中,固化不提供 在防止方面额外改善表面质量 非特异性吸附生物分子。
    5. 质量控制: 胺基浓度。你可以测量的浓度 胺基团在盖玻片表面上使用TRIONE茚三酮试剂。至 这个结束,通过将一个盖玻片放入一个50毫升Falcon管和 ?在摇摆式离心机中以2,000×g离心1分钟。然后 填充管1毫升茚三酮试剂,并按照 制造商的方案用分光光度法量化平均值 盖玻片表面上的胺基密度(对于24×40mm 盖玻片,厚度?0.15 mm,总表面积为 1,940mm 2/sup)。使用标准稀释的APTES作为校准 曲线,我们通常每nm 2个获得3个胺基。

  4. 在浊点条件下用NHS-PEG处理胺 - 玻璃表面
    注意:
    1. 该方法基于Ha等人介绍的方法。 (2002), 其中修饰使PEG分子的密度最大化 阻断和中和PEG之后的带正电荷的胺基团 沉积(E部分)。因此,胺处理的盖玻片被偶联 琥珀酰亚胺基-PEG在pH 9.0下在含有0.45M的碳酸氢盐缓冲液中 在10.0%NHS-PEG(w/v)中的K 2 SO 4 SO 4。高盐和高PEG 浓度使PEG溶液刚好低于其"浊点",其中 ?使PEG层在表面上的密度最大化(Kingshott et et al。,2002)。
    2. NHS基团在水性缓冲液中水解,这使得 NHS-PEG非反应性。 NHS组的寿命在几个之间 秒和几分钟,这取决于NHS-PEG的性质 接头,pH和温度(Hermanson,2008)。例如,我们发现 NHS-SVA在室温下在pH9.0下半衰期为?5分钟水解 温度,这是反应性之间的良好折衷 NHS基团和反应物种寿命。因此,我们的协议 最小化反应性NHS-SVA物种在pH = 9.0下花费的时间 以添加到胺 - 玻璃表面。具体来说,我们最初 溶解干燥的PEG-SVA,pH6.0,NHS基团保留 未水解至少1小时。然后,在即将添加之前 PEG溶液到玻璃表面,我们使pH达到9.0。如果你是 ?使用非SVA接头,必须优化反应的pH 达到NHS的使用寿命?5分钟。
    3. 反应的寿命 ?NHS基团可以通过分光光度法测定 游离NHS在水解时的积累动力学[游离NHS强烈 在260nm处吸附(Miron,1982)]。我们也强烈推荐测量 ?在新批购买的PEG中的反应性PEG-NHS的百分比 试剂。我们已经有完全水解,非反应性的情况 ?PEG已由主要供应商装运。
    1. 该步骤最好在步骤C11的APTES孵育过程中进行。取出 ?6的一次性使用等分的干mPEG-SVA从-80℃储存。每 等分试样应为约5mg,其足以治疗两个 盖玻片。 6个等分试样中干燥的mPEG-SVA的质量应该是 在每个管上预先写入0.1mg精确度,然后在-80℃下储存 ?(对于6个管,例如4.9,5.0,5,1,4.9,5.0和5.1mg)。此外, 从储存中取出1-2mg生物素-PEG-SVA。让所有的管暖和 ?室内温度。
    2. 此步骤最好在APTES期间完成 在步骤C11孵育。计算0.5M K 2 SO 4溶液的体积 通过将等分试样质量乘以8来添加到每个mPEG-SVA管中(例如 ?含有4.9mg的管将需要4.9×8 =39.2μlK 2 SO 4溶液),并将所有体积记录在笔记本中。总结所有的卷, 以获得制备六个PEG所需的最小体积为0.5M K 2 SO 4 SO 4 溶液[例如],用于6个包含4.9,5.0,5,1,4.9,5.0和5.1的管 ?mg mPEG-SVA需要(4.9 + 5.0 + 5.1 + 4.9 + 5.0 + 5.1)×8 =240μl 的K 2 SO 4子]。基于计算的最小体积,准备足够 量的在0.5M K 2 SO 4中的0.5%生物素-PEG-SVA溶液(例如溶解1.3 ?mg具有260μl0.5M K 2 SO 4的生物素-PEG-SVA)。吸取预先计算 ?体积的0.5%生物素-PEG-SVA溶液加入到每个6mPEG-SVA中 ?等分试样(例如向4.9mg管中加入39.2μl,等等),涡旋 10秒,并用台式离心机短暂旋转。每个管现在 应该有一个清楚的溶液和一个小(?5微升体积)PEG的丸 ?在底部。
      注意:PEG沉淀是预期的,因为 PEG溶液目前为11.6%浓度(w/v),这是以上 其"浊点"(10%w/v)。
      管可以留在房间 温度至少1小时,没有明显的NHS水解 组,而盖玻片正在用APTES治疗和布局 ?PEG化。
    3. 所有APTES处理的盖玻片布局 PEG化,使用一个PEG-NHS等分试样建立PEG化反应 ?一个时间。为此,向PEG溶液中加入1/8的1M NaHCO 3(pH 9.0) ?在原始包含的管中加入4.9μlNaHCO 3(3mL),并在37℃下加入 ?一个4.9mg mPEG-SVA等分试样,现在含有39.2μl 生物素-PEG-SVA溶液),通过上下吹吸快速混合,和 将整个溶液沉积在干燥的APTES处理的中心 盖玻片。用另一个盖玻片仔细盖住滴,确保 两个盖玻片的刻痕表面面向PEG 溶液,并避免气泡的形成(通过实践)。重复 剩余5对盖玻片并在室温下孵育30分钟 温度 注意:加入后,PEG溶液看起来清澈 NaHCO ,因为总PEG浓度现在刚好低于' ?点'。我们注意到,用新鲜的盖玻片的额外涂层 PEG溶液不能改善表面的质量(就其而言) 生物分子的非特异性吸附)。
    4. 小心分开 盖玻片对使用镊子和放置单个盖玻璃回 进入陶瓷架在双蒸水中。冲洗广泛 用水直至溶液不再发泡。
      质量 control:PEG分子的密度。您可以估计包装密度 的PEG分子在玻璃表面上沉积 荧光素-PEG-NHS在与上述相同的条件下,和 使用高灵敏度双光束测量494nm处的吸光度 分光光度计(例如Perkin Elmer Lambda 35)。分子密度= ?[(OD 494 - 消光系数)* 6.02×10 23薄膜/支持体] /面积。这样 估计,平均封装密度应与之一致 在浊点[R = 2.8nm,M w w = 5000 PEG分子的回转半径 (Dalsin等人,2005)]。

  5. 用乙酰基封闭未反应的胺
    注意:由于PEG分子的庞大性,表面上只有一部分胺基团与NHS-PEG反应。结果,剩余的胺有助于带负电荷的分子例如DNA的非特异性吸附,这在低离子强度的缓冲液中是有问题的。因此,我们用NHS-乙酸酯封闭未反应的胺,将其转化成中性,稳定的酰胺。
    1. 如步骤C12中所述吹干12聚乙二醇化盖玻片。放置干燥 盖玻片上,聚乙二醇化表面朝上。
    2. 在使用前,立即溶解3毫克磺基-NHS-乙酸盐在300微升 的0.1M NaHCO 3(pH 9.0),将50μl的溶液沉积在6孔板上 ?PEG盖玻片,并用剩余的6个盖玻片覆盖 PEG化表面面向溶液。在室温下孵育30分钟 温度。用双蒸水冲洗盖玻片,吹干,和 ?在-80°C下保存干燥。

  6. 测试PEG化表面的荧光背景和生物分子的非特异性吸收 注意:以下方案用于快速测试表面对荧光标记的生物分子的非特异性"粘性"。然而,该测试不涉及表面固定化分子的生物活性(例如,表面固定化DNA上的转录因子的活性)。因此,表面质量的最相关的测试是表面结合的生物分子的活性的生物化学测定,其与阳性对照并行进行,其中在基于溶液的条件下测量相同量的生物分子的活性Revyakin等,2012)。
    1. 使用1毫米宽的条带在盖玻片上创建5个开放孔 双面胶带(通过简单地将6个条带粘贴到改性面上), ?并将盖玻片放置在目标型TIRF显微镜上。 5 孔允许测试5种不同的生物分子。
    2. 存放5微升 ?滴入PBST进入井,打开激发激光,并聚焦 显微镜到表面上。获取电影以测量荧光 背景在表面,在单分子成像条件下 (例如,使用532和640nm光源在密度?0.5kW/cm 2下的激发) 成像在580/60nm和670/45nm光带,采样率2.5Hz)。 ?在我们手中,硅烷化和聚乙二醇化增加的数量 与Piranha处理相比,荧光背景斑点?3倍 玻璃( 从约3个点增加到约10个点/100x100μm) 视野),这是大多数背景的可接受水平 单分子成像实验。允许背景点 photobleach。
    3. 准备10nM的荧光溶液 在PBST中标记的测试蛋白,并在顶部沉积20μl的溶液 的5μlPBST液滴已经在孔中。获取电影进行量化 分子对表面的非特异性吸附。对于 简单,在这个测试我们不提供氧清除剂 测试蛋白溶液。因此,避免立即光漂白 分子粘附到表面上(这将导致 过度估计表面质量)使用最小激光功率(30?100 W/cm 2 )和在5秒内光漂白的荧光标记 没有氧清除剂(例如Cy3和Atto633)。为了质量好 表面,我们通常观察到10nM生物分子的"云"迅速 在本体溶液中扩散(以2.5Hz的采集速率),和?10 在任何给定的电影帧(100×100μm)中的单分子有限斑点 视场)。不应观察到斑点的额外积累 在10分钟内(激发光关闭,然后转动 再次),表明非特异性吸附是罕见的 可逆。用其他感兴趣的生物分子重复测试 其余4口井。

食谱

  1. 0.5 M KOH(每升)
    28克KOH
    注意:解决方案可以重复使用。
  2. 0.5M K sub SO 4(pH 6.0)(每升)
    174g K sub 2 SO 4 4/
    注意:存放在室温下。如果您的双蒸水供应为酸性,则无需调整K 2 SO 4 解决方案。如果您的双蒸水供应是基本的,请使用稀释的H 2 SO 4
  3. 1摩尔NaHCO 3(pH 9.0)(10x)(每升)
    82g NaHCO 3/v/v 注意:用NaOH调节pH至9.0,并以一次性使用的等分试样储存在-80℃。
  4. 含有0.1%吐温(PBST)(每升)的磷酸盐缓冲盐水
    2.4g KH 2 PO 4 4/
    80克NaCl
    14.4g Na 2 HPO 4
    1ml吐温20

致谢

我们感谢集团的史蒂文·朱和罗伯特Tjian的支持(加州大学伯克利分校和Janelia农场研究Camus,由NIH资助1P01CA112181-01A1和霍华德休斯医学研究所支持)。

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Copyright Zhang et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhang, Z., Park, S. R., Pertsinidis, A. and Revyakin, A. (2016). Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy. Bio-protocol 6(7): e1784. DOI: 10.21769/BioProtoc.1784.
  2. Zhang, Z., Revyakin, A., Grimm, J. B., Lavis, L. D. and Tjian, R. (2014). Single-molecule tracking of the transcription cycle by sub-second RNA detection. Elife 3: e01775.
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