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Sep 2016
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Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells
Fab片段结合荧光染料用于活细胞单分子示踪   

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Abstract

Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have become valuable tools to discern how endogenous proteins control cell biology. Yet, some technical challenges remain; in SMT, these revolve around the characteristics of the labeling reagent. A good reagent should have neutrality (in terms of not affecting the target protein’s functions), tagging specificity, and a bright fluorescence signal. In addition, a long shelf-life is desirable due to the time and monetary costs associated with reagent preparation. Semiconductor-based quantum dots (Qdots) or Janelia Fluor (JF) dyes are bright and photostable, and are thus excellent candidates for SMT tagging. Neutral, high-affinity antibodies can selectively bind to target proteins. However, the bivalency of antibodies can cause simultaneous binding to two proteins, and this bridging effect can alter protein functions and behaviors. Bivalency can be avoided using monovalent Fab fragments generated by enzymatic digestion of neutral antibodies. However, conjugation of a Fab with a dye using the chemical cross-linking agent SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) requires reduction of the interchain disulfide bond within the Fab fragment, which can decrease the structural stability of the Fab and weaken its antigen-binding capability. To overcome this problem, we perform limited reduction of F(ab’)2 to generate Fab’ fragments using a weak reducer, cysteamine, which yields free sulfhydryl groups in the hinge region, while the interchain disulfide bond in Fab’ is intact. Here, we describe a method that generates Fab’ with high yield from two isoforms of IgG and conjugates the Fab’ fragments with Qdots. This conjugation scheme can be applied easily to other types of dyes with similar chemical characteristics.

Keywords: Single-molecule tracking (单分子示踪), Live-cell imaging (活细胞成像), Antibody (抗体), Quantum dots (Qdots) (量子点), Janelia Fluor (JF) dyes (Janelia Fluor (JF)染色), Fab’ (Fab’), Fab (Fab), Conjugation (共轭), Cysteamine, Pepsin (胃蛋白酶)

Background

Cell-surface protein functions are tightly regulated in their native environment. Gaining a comprehensive understanding of their functions necessitates monitoring their interactions with various cell membrane components, such as other proteins and lipids, and cytoskeletal machinery and cellular organelles below the membranes. Optical tools enabling live-cell based imaging at a molecular level (Joo et al., 2008; Lord et al., 2010; Chung, 2017) include conventional methods such as confocal microscopy and total internal reflection fluorescence microscopy (TIRFM) combined with specific modalities such as Förster resonance energy transfer (FRET) (Sekar and Periasamy, 2003), single-molecule tracking (SMT) (Moerner, 2012), and fluorescence correlation spectroscopy (FCS) (Kim et al., 2007), and cutting-edge super-resolution microscopy, such as photoactivated localization microscopy (PALM) (Betzig et al., 2006), stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006), stimulated emission depletion (STED) microscopy (Hein et al., 2008), structured illumination microscopy (SIM) (Gustafsson, 2000 and Gustafsson et al., 2009), and lattice light sheet (LLS) microscopy (Chen et al., 2014). Using these tools for live-cell imaging to monitor endogenous proteins requires bright fluorophore-coupled reagents that specifically bind to target proteins. To this end, bright dyes such as semiconductor quantum dots (Qdots) (Dahan et al., 2003; Chung and Bawendi, 2004; Lidke et al., 2004; Chung et al., 2010; Bien-Ly et al., 2014; Chung and Mellman, 2015; Chung et al., 2016) and Janelia Fluor (JF) dyes (Grimm et al., 2015 and 2017) conjugated to high-affinity and non-perturbing antibody-based reagents are widely used. However, an antibody can bind to two target proteins simultaneously. This problem is typically circumvented by digesting antibodies to Fab fragments using proteolytic enzymes such as papain, which cleaves at the hinge region of immunoglobulins (Chung et al., 2010; Bien-Ly et al., 2014; Chung and Mellman, 2015; Chung et al., 2016). Conjugation of Fab with fluorescent dyes relies on a thiol-maleimide reaction. This reaction, however, can destabilize Fab when the interchain disulfide bond within a Fab is reduced, which elicits loss of binding capability to target proteins within a relatively short period of time (a few weeks at best). Consequently, laboratories must frequently regenerate the conjugates, imposing higher cost and hours lost. Thus, we use Fab’ fragments containing free sulfhydryl groups in the hinge region (Selis et al., 2016), which can be used for a succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)-based conjugation reaction without reducing the interchain disulfide bond within Fab’. To this end, we perform two-step reactions, in which IgG is digested into F(ab’)2 by pepsin and Fab’ is generated by limited reduction of F(ab’)2 using cysteamine. In this protocol, we showcase a method that generates Fab’ fragments from two different types of antibodies and subsequently conjugates one type of Fab’ with quantum dots (Qdots) to monitor EGFR on live cells using SMT. We believe this conjugation scheme will most likely improve the overall yield and stability of the tagging reagents for various types of live-cell imaging of endogenous proteins.

Materials and Reagents

  1. Pipette tips (Olympus, catalog numbers: 24-120RL, 24-150RL, 24-165RL)
  2. Sterile pipette tips (Olympus, catalog numbers: 24-401, 24-404, 24-412, 24-430)
  3. Sterile serological pipets (Olympus, catalog numbers: 12-102, 12-104)
  4. 15 ml centrifuge tubes (Olympus, catalog number: 28-101)
  5. 50 ml centrifuge tubes (Fisher Scientific, catalog number: 14-955-239)
  6. 1.5 ml microcentrifuge tubes (Olympus, catalog number: 24-281)
  7. 1.5 ml Protein LoBind Tubes (Eppendorf, catalog number: 022431081)
  8. 15 ml Protein LoBind Conical Tubes (Eppendorf, catalog number: 0030122216)
  9. Adjustable-volume pipettes (Eppendorf, catalog number: 2231300008)
  10. Pierce disposable columns (Thermo Scientific, catalog number: 29920)
  11. NAP-5 desalting columns (GE Healthcare, catalog number: 17-0853-01)
  12. µ-Dish 35 mm, high glass bottom (Ibidi, catalog number: 81158)
  13. Treated cell culture flasks (Thermo Scientific, catalog number: 12-556-010)
  14. Pierce Protein Concentrators PES, 30K MWCO, 0.5 ml (Thermo Scientific, catalog number: 88502)
  15. Pepsin (Sigma Aldrich, catalog number: P6887-250MG)
  16. Mouse IgG1 Isotype Control (Invitrogen, catalog number: 02-6100); αEGFR (rat IgG2a) antibody (Abcam, catalog number: ab231)
  17. MDA-MB-468 breast cancer cell line (ATCC, catalog number: HTB-132)
  18. BCA protein assay kit (Thermo Scientific, catalog number: 23225)
  19. Superdex G200 (GE Healthcare, catalog number: 17-1043-01)
  20. Sulfo-SMCC [sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate], No-Weigh Format (Thermo Scientific, catalog number: A39268)
  21. DMSO (Dimethylsulfoxide) (Thermo Scientific, catalog number: 20684)
  22. Qdot 605 ITK Amino (PEG) Quantum Dots (amino-PEG-Qdot605, Invitrogen, catalog number: Q21501MP) or Qdot 565 ITK Amino (PEG) Quantum Dots (amino-PEG-QDot565, Invitrogen, catalog number: Q21531MP)
  23. HEPES, 1 M solution, pH 7.3, molecular biology grade, ultrapure (Thermo Scientific, catalog number: J16924AE)
  24. Sodium chloride, 5 M (Lonza, catalog number: 51202)
  25. Cysteamine (Sigma-Aldrich, catalog number: M9768-5G)
  26. Dye labeled marker, CAL Fluor Red 610 T10 (LGC Biosearch Technologies, catalog number: RD-5082-5)
  27. Glycerol (Thermo Scientific, catalog number: J16374AP)
  28. Dulbecco's PBS (GenClone, catalog number: 25-508)
  29. Trypsin EDTA (Corning, catalog number: 25-052-CV)
  30. Pierce F(ab’)2 Preparation Kit (Thermo Scientific, catalog number: 44988, see Note 1), include:
    1. Zeba Spin Desalting Columns (Thermo Scientific, catalog number: 89889)
    2. NAb Protein A Plus Spin Columns (Thermo Scientific, catalog number: 89956)
    3. PBS (Thermo Scientific, catalog number: 1890535)
  31. Acetate buffer, pH 4.0 (Fisher Scientific, catalog number: 50-255-309)
  32. EDTA (Fisher Scientific, catalog number: 03-500-506)
  33. Cell growth medium (RPMI 1640 with 10% fetal bovine serum and 1% penicillin-streptomycin)
    1. RPMI 1640, with L-Glutamine, 2000 mg/L D-Glucose (GenClone, catalog number: 25-506)
    2. Fetal bovine serum (FBS), heat-inactivated, U.S. Origin (GenClone, catalog number: 25-514H)
    3. Penicillin-streptomycin mixture (Lonza, catalog number: 17-602E)
  34. Acetate digestion buffer, pH 4.0 (see Recipes)
  35. Exchange buffer, pH 7.2 (see Recipes)

Equipment

  1. Eppendorf easypet 3 (Eppendorf, catalog number: 4430000018)
  2. pH meter (Fisherbrand, Accumet, model: 15)
  3. Vortex mixer (VWR, model: Analog Vortex Mixer)
  4. Centrifuge (Eppendorf, model: 5810R)
  5. Thermo mixer (Thermo Scientific, model: 13687720)
  6. End-over-end mixer (Argos Technologies, RotoFlex, model: R2000)
  7. CO2 incubator air jacket TC (VWR, catalog number: 10810-902)
  8. Biosafety cabinet (LabConco, model: A2)
  9. Nikon Eclipse TE2000 inverted microscope with TIRF illuminator and a 100x/1.49NA Plan Apo objective (Nikon, model: Eclipse TE2000-E)
  10. iXon back-illuminated EMCCD camera (Andor Technology, catalog number: DU-888E-C00-#BV-500)
  11. 488 nm line of solid-state lasers (Andor Technology)

Software

  1. ImageJ 1.52i with Java 1.8.0_172

Procedure

This procedure describes generation of Fab’ fragments from IgG and their subsequent conjugation to fluorescent dyes (here, we use Qdot), as summarized in Figure 1. We optimized the digestion and reduction schemes using a mouse IgG1, and then applied these procedures (see Note 8) to further optimize the conditions for conjugating Fab’ fragments of an αEGFR antibody (rat IgG2a) with Qdots to perform SMT.


Figure 1. Overview of the procedure with cartoon schematic. The reaction conditions may vary between different antibodies (see Notes 5 and 11).


  1. Preparation of acetate digestion buffer and pepsin solution
    1. Prepare acetate digestion buffer (Recipe 1).
    2. Prepare a 10 mg/ml pepsin solution in acetate digestion buffer (see Notes 2 and 3).

  2. IgG sample preparation (see Note 4)
    1. Twist off the bottom closure of a Zeba Spin Desalting Column and loosen cap. Place the column in a 15 ml centrifuge tube.
    2. Centrifuge the column at 1,000 x g for 2 min to remove storage solution. Discard the flow-through.
    3. Add 1 ml of acetate digestion buffer to the column. Centrifuge at 1,000 x g for 2 min and remove the flow-through. Repeat this step three times.
    4. Place the equilibrated column in a 15 ml Protein LoBind Conical Tube. Remove the cap and slowly add 0.5 ml of antibody sample to the center of the resin bed in the column. Be careful not to disturb the resin bed.
    5. Replace the cap and centrifuge at 1,000 x g for 2 min. Collect the IgG sample. Figure 2 shows that the total IgG amount was slightly decreased (Lane 2) relative to the original amount (Lane 1) from this buffer exchange step.

  3. Pepsin digestion of IgG into F(ab’)2
    1. Transfer 0.5 ml of the prepared IgG sample to a 1.5 ml Protein LoBind Conical Tube.
    2. For 1 mg/ml IgG, add 2.5 μl of 10 mg/ml pepsin solution to the tube. Antibody/pepsin w/w ratio is 20:1.
    3. Let the digestion reaction proceed for 16 h on a thermo mixer at 37 °C with agitation at 1,000 rpm. This condition can vary for different IgG molecules (see Note 5). The band of ~100 kDa in lane 3 in Figure 2 shows F(ab’)2.

  4. Termination of the digestion reaction (see Note 6)
    1. Twist off the bottom closure of a Zeba Spin Desalting Column and loosen the cap. Place the column in a 15 ml centrifuge tube.
    2. Centrifuge the column at 1,000 x g for 2 min to remove storage solution.
    3. Add 1 ml of PBS (pH 7.4) to the column. Centrifuge at 1,000 x g for 2 min and discard the flow-through. Repeat this step three times.
    4. Place the equilibrated column in a 15 ml Protein LoBind Conical Tube. Remove the cap and slowly apply 0.5 ml of the digested IgG sample to the center of the resin bed. Be careful not to disturb the resin bed. Pepsin digestion of IgG will be terminated at this step.
    5. Replace the cap and centrifuge at 1,000 x g for 2 min to collect the flow-through. See Lane 4 in Figure 2.

  5. Purification of F(ab’)2 sample
    1. Allow the NAb Protein A Plus Spin Column and PBS to come to room temperature. Set the centrifuge speed to 1,000 x g.
    2. Loosen the top cap on the NAb Protein A Plus Spin Column and snap off the bottom closure. Place the column in a 15 ml centrifuge tube and centrifuge for 1 min to remove storage solution. 
    3. Disperse the resin by adding 2 ml of PBS. For a 0.5 ml sample, use half of the resin. 
    4. Equilibrate the resin in the column with 2 ml of PBS. Centrifuge for 1 min and discard the flow-through. Repeat this step once.
    5. Cap the bottom of the column with the included rubber cap. Apply the digestion mixture to the column and cap the top tightly. Resuspend the resin and sample by inversion. Incubate at room temperature on an end-over-end mixer for 15 min.
    6. Loosen the top cap and remove the bottom cap. Place the column in a 15 ml Protein LoBind Conical Tube and centrifuge for 1 min. Save the flow-through (this fraction contains F(ab’)2). See Lane 5 in Figure 2.
    7. For optimal recovery, wash the column with 0.5 ml PBS. Centrifuge for 1 min and collect the flow-through. Repeat and collect two wash fractions containing additional F(ab’)2 (see Lanes 6 and 7 in Figure 2 and Notes 7 and 8).
    8. Measure protein concentration using BCA protein assay.
      1. Prepare the BCA reagents and a serial dilution of the protein standard following the manufacturer’s instruction. Mix 200 μl of the BCA reagents with 10 μl of either the standards or the samples collected above. 
      2. After incubation at 37 °C for 30 min, the color of the mixture will turn from pale green to purple in response to the protein concentration. See Figure 3. 
      3. Measure the absorbance at 562 nm with a spectrophotometer or a plate reader to estimate the protein concentration by comparison to the standard curve.


        Figure 2. SDS-PAGE analysis of samples from each step before and after pepsin digestion of mouse IgG1. Lane 1: The whole IgG1 sample that was originally stored in PBS (pH 7.4). Lane 2: The IgG1 sample after a buffer exchange to bring it to the acetate digestion buffer (pH 4.0). Lane 3: The sample resulting from a 16 h digestion on a thermo shaker at 37 °C (a strong band at ~100 kDa position indicates F(ab’)2). Lane 4: The sample after a buffer exchange to bring it to PBS (pH 7.4) to terminate the reaction. Lane 5: The flow-through from a Protein A column after a 15-min incubation at room temperature. Lanes 6 and 7: Two consecutive portions collected from washing the protein A column to maximize the output.


        Figure 3. BCA assay for determining the protein concentrations after purification. A. The degree of green-to-purple color conversion of BCA solutions is proportional to the protein concentration. B. A standard curve of absorbance (at 562 nm) versus known protein concentrations (y = 0.507x + 0.0188) is used to determine the concentrations of F(ab')2 samples collected from the Protein A column flow-through.

  6. Preparation of the separation column for Fab’-Qdot conjugate
    Note: This segment is a modification of the Qdot® Antibody Conjugation protocol by Invitrogen.
    1. Suspend the separation medium (Superdex G200) in the bottle by gentle shaking or vortexing. Ensure the medium is fully suspended before starting column preparation with Pierce disposable columns.
    2. Mark the column with two lines, one at 45 mm above the top of the frit, and a second at 55 mm above the frit (Figure 4A).
    3. Wet the frit with pure water before loading the media. After ensuring that the separation medium is a uniform suspension, load media into the column with a 1 ml pipette to the second line at the 55 mm mark (Figure 4B) and let it settle into a packed gel bed that is ~45 mm high (Figure 4C).
    4. Gently add 0.5 ml distilled water to the top of the gel while maintaining a level bed surface. 
    5. As the solvent level drops to near the top of the settled gel bed, fill the column with PBS, and allow the PBS level to drip down to just above the top of the gel bed. Repeat this two more times.
    6. Replace the bottom and the top caps when the PBS level from the last fill drops to 2 to 3 mm above the top of the settled gel bed.


      Figure 4. Preparation of the separation column. A. The image shows two marks (45 and 55 mm) above the frit. B. The gel suspension fills the column to the upper mark (55 mm). C. The gel settles to the lower mark (45 mm).

  7. Activation of Qdot nanocrystals (see Note 9)
    1. Prepare a freshly dissolved 20 mM solution of sulfo-SMCC in DMSO. To do this, dissolve 2 mg Pierce No-Weigh Sulfo-SMCC in 229 μl of DMSO.
    2. Add 1.75 μl of 20 mM solution of sulfo-SMCC in DMSO to 62.5 μl of an 8 μM stock solution of amino-PEG-QDot605. Vortex briefly.
    3. Incubate for 1 h at room temperature with agitation at 500 rpm to activate the Qdots. Avoid light.
    4. Prepare desalting columns while the activation step is proceeding.

  8. Equilibration of desalting columns
    1. Prepare exchange buffer (see Recipe 2).
    2. Label the NAP-5 desalting columns. Mark one with “reduced Fab’” and the other with “activated Qdot”.
    3. Remove the top and bottom caps from both columns and allow the storage liquid in the columns to drain. Just as the liquid in each column is approaching the top of the column gel bed, begin adding exchange buffer.
    4. Equilibrate each column with 10 ml of exchange buffer.
    5. While there is still exchange buffer visible above the gel bed in each column, cap the bottom of each column and set that aside until the F(ab’)2 reduction and activation of Qdot are completed.

  9. Generation of Fab’ fragments
    1. Prepare 100 μg of F(ab’)2 in 300 μl of PBS by dilution or concentration, as necessary.
    2. When the Qdot activation is almost finished, incubate the F(ab’)2 sample with cysteamine (5 mM) and EDTA (2 mM) at 10 °C for 5 min, mixing them at 500 rpm (see Note 8 and 10). The bands in Lane 2 of Figure 5 represent Fab’ (50 kDa) and further reduced fragments (25 kDa); the latter fragments are unlikely to retain antigen binding capacity. To maximize Fab’ yield, we recommend testing various reduction conditions (see Notes 11 and 12) for different F(ab’)2 samples using small aliquots (~5-10 μl each lane) for gel analysis.


      Figure 5. SDS-PAGE analysis of F(ab’)2 reduction to Fab’ with cysteamine. Lane 1: The F(ab’)2 sample from Lane 5 in Figure 2. Lane 2: The sample after a reduction with 5 mM of cysteamine for 5 min at pH 7 and 10 °C (see Note 11), before buffer exchange. The band at ~50 kDa indicates Fab’.

  10. Desalting and collection of the reduced Fab’ fragment
    1. Add 40 μl of distilled water to one vial of the dye-labeled marker and mix. This makes enough dye-labeled marker for two conjugation reactions. Store at 2-6 °C when not in use.
    2. Add 500 μl of water to a 1.5 ml microcentrifuge tube and mark the outside of the tube at the meniscus. Add another 500 μl of water and make a second mark on the outside of the tube corresponding to a 1,000 μl volume. Discard the water. This tube is used to collect the reduced Fab’ in Step J7 and the activated Qdot nanocrystals in Step K4.
    3. When the F(ab’)2 reduction is completed (Step I2), add 20 μl of dye-labeled marker (prepared in Step J1) to the reduced Fab’.
    4. Uncap the desalting column labeled “reduced Fab’” and allow the remaining exchange buffer to enter the gel bed. Immediately following this, add the reduced mixture (prepared in Step J3) to the top of the gel bed.
    5. Allow the reduced Fab’ mixture to completely enter the gel.
    6. Add 1 ml of exchange buffer to the top of the gel bed to elute the Fab’.
    7. Begin collecting reduced Fab’ into a centrifugation tube (marked in Step J2) when the first colored drop elutes (see Figure 6); collect no more than 500 μl (to the lower marked line).


      Figure 6. Collection of the reduced Fab’ fragment from the desalting column. A. One milliliter of exchange buffer was added to the desalting column after the mixture of the reduced Fab’ with the dye marker entered the column completely. B. The first stained drop is being collected in the tube marked in Step J2.

  11. Desalting and collecting the activated Qdot nanocrystals
    1. Uncap the desalting column labeled “activated Qdot”. Allow remaining exchange buffer to enter the gel bed. Immediately after this, add the activated Qdot nanocrystals (from Step G3) to the top of the gel bed.
    2. Allow the activated Qdot nanocrystals mixture to completely enter the gel bed.
    3. Add 1 ml of exchange buffer to the top of the gel bed to elute the Qdot nanocrystals.
    4. When the first drop of colored material elutes from the column, begin collecting directly into the centrifugation tube containing the reduced and desalted Fab’. See Figure 7.
    5. Stop collecting when the final volume reaches 1 ml (up to the top line marked in Step J2; 500 μl of activated Qdot nanocrystals).
    6. Mix briefly.


      Figure 7. Collection of the activated Qdot from the desalting column. A. One milliliter of exchange buffer was added to the desalting column after the activated Qdots completely entered the gel. B. The first stained drop is being collected in a tube.

  12. Conjugation reaction
    Allow the reduced Fab’ and activated Qdot nanocrystals to react for 2 h at room temperature. Avoid light.

  13. Concentrating the sample
    1. Split the volume (from Step L1) into two protein concentrators.
    2. Concentrate each volume to ~20 μl by centrifuging at 4,000 x g for 15 min. If the volume is > 20 μl after the centrifugation, continue centrifuging for another 5 min.

  14. Separation of the Qdot-conjugated Fab’ from unconjugated Fab’
    1. Uncap the separation column (from Step F6) and allow the remaining PBS to enter the gel bed by gravity. Immediately following this, add the concentrated conjugate reaction solution combined from the two protein concentrators (~40 μl total volume) to the top of the column. Avoid disturbing the gel bed.
    2. Allow the conjugate reaction solution to enter the gel and then gently add 50 μl PBS. Let this buffer run into the gel bed.
    3. Gently fill the reservoir above the column with PBS and allow the sample to elute by gravity. Visually monitor the “dead space” between the frit and the column tip.
    4. When color appears in the “dead space,” collect only the first 8-10 drops of colored solution in a centrifugation tube. See Figure 8. 
    5. Determine the Qdot concentration by absorbance measurements at the first absorption peak of the Qdot.
    6. Bring the final conjugate pool to 50% v/v of glycerol and store at 4 °C.


      Figure 8. Collection of the Qdot-conjugated Fab’ from the separation column. A. The separation column is filled with PBS after the conjugation mixture completely enters the column. B. The collection of the eluate begins when the color appears in the dead space.

  15. Single-molecule tracking of EGFR on live cells using αEGFR Fab’-Qdot605 conjugates
    1. Digest αEGFR antibodies into F(ab’)2 using pepsin [20:1 antibody/pepsin (w/w ratio)] in acetate digestion buffer (pH 4.0) at 37 °C for 16 h with agitation, followed by a Protein A column purification (Figure 9A).
    2. Reduce the resulting F(ab')2 to Fab' with 5 mM cysteamine at 10 °C for 5 min (Figure 9B).
    3. Conjugate Fab’ with activated Qdot605 for generating αEGFR Fab’-Qdot605 conjugates.
    4. Allow 2 nM conjugates in the full growth medium to bind to target proteins (EGFR) for 10 min at room temperature on live cells (MDA-MB-468 breast cancer cell line that overexpresses EGFR) plated on glass bottom dishes. Wash three times with full growth medium (See Note 13).
    5. Perform single-molecule tracking (SMT) (we followed the methods described in Chung and Mellman, 2015; Chung, 2017) with light excitation at 488 nm using the TIRFM on an inverted microscope with a 100x/1.49NA Plan Apo objective. Image acquisition was done at ~11 Hz using an EMCCD camera. One snapshot image of individually labeled Qdots that was rendered using ImageJ is shown in Figure 9C.
    6. Estimate the binding specificity of the conjugates by determining the density of bound conjugates after saturating the epitope binding with original antibody (~100 times the Kd will suffice), relative to the density without the saturation. The binding specificity of the Fab’-QD conjugates was ~89%.


      Figure 9. Generation of Fab’-Qdot conjugates for SMT using αEGFR antibodies (IgG2a). A and B. An αEGFR IgG2a was used to demonstrate the applicability of the Fab’-Qdot605 conjugates in SMT on live cells. SDS-PAGE analyses of the pepsin digestion (A) and cysteamine reduction (B) of αEGFR rat IgG2a. (B) The Fab’ throughput of the IgG from the reduction showed similar dependencies to changes of temperature, cysteamine concentration, and pH to those for the mouse IgG1 F(ab’)2 shown in Figure 5 (see Note 11). C. One snapshot (10.72 Hz) SMT image (488 nm illumination, 100x oil objective) by a total internal reflection fluorescence microscope (TIRFM) after labeling EGFR with the αEGFR Fab’-Qdot605 conjugates on ~ 60% confluent MDA-MB-468 cells.

Notes

  1. Procedures A-E are modified from the protocol for Pierce F(ab’)2 Preparation Kit (Thermo Scientific, catalog number: 44988). Zeba Spin Desalting Columns, PBS, and NAb Protein A Plus Spin Columns used in this protocol can be found in this kit.
  2. Pepsin solution should be prepared freshly for each reaction.
  3. Pepsin will be irreversibly denatured in higher pH buffer. Never prepare the pepsin solution in neutral buffer.
  4. When using concentrated IgG samples, simply dilute the samples to desired concentration in acetate digestion buffer and skip Procedure B.
  5. Pepsin digestion varies for different IgG molecules. A time course (1-5 and 16 h) test using small aliquots of desired IgG before the preparation of F(ab’)2 is recommended. If the digestion time is set to be 16 h as proposed in Procedure C, start this reaction at the end of a day and stop the reaction the next morning.
  6. The purpose of changing the acetate buffer to PBS after the pepsin digestion is to terminate the reaction and to prepare the sample for Protein A purification (for removal of undigested IgG) since the Protein A column is ineffective at acidic pH.
  7. Protein A column can be regenerated by following the manufacturer’s instructions using IgG elution buffer (pH 2.8).
  8. The digestion/reduction products can be verified using non-reducing SDS polyacrylamide gel electrophoresis as shown in Figures 2, 5, and 9, as necessary.
  9. This conjugation scheme can be applied directly to a maleimide linked dye, where active maleimide can react with sulfhydryl groups within the Fab’. 
  10. Cysteamine is sensitive to air and moisture. Store the original bottle in a vacuum desiccator. Prepare cysteamine solution right before use.
  11. The reduction yield by cysteamine may vary depending on different F(ab’)2 samples. A pilot test on a range of pH (5, 6, and 7), temperature (4 °C, 10 °C, 22 °C, and 37 °C), and cysteamine concentration (0.5-20 mM) is highly recommended.
  12. HPLC or FPLC can be used to further purify the reduced sample for Fab’. 
  13. The incubation time and concentration of the conjugates can vary depending on experiments, affinity of the Fab’, etc., We typically vary these in the ranges of 2-10 min (incubation time) and 0.5 to 10 nM (conjugate concentration).

Recipes

  1. Acetate digestion buffer, pH 4.0
    0.1 M acetate buffer
    0.01 M EDTA
  2. Exchange buffer, pH 7.2
    50 mM HEPES
    150 mM NaCl

Acknowledgments

We thank the members of the Chung Laboratory for useful discussion and Jore Kotryna Vismante for photographing the procedure. This work was supported by the GW Cancer Center and Katzen Research Cancer Research Pilot Award. The procedure introduced here was modified from past single-molecule tracking studies (Chung et al., 2010; Bien-Ly et al., 2014; Chung et al., 2016).

Competing interests

The authors declare no financial or non-financial competing interests related to this work.

References

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  2. Bien-Ly, N., Yu, Y. J., Bumbaca, D., Elstrott, J., Boswell, C. A., Zhang, Y., Luk, W., Lu, Y., Dennis, M. S., Weimer, R. M., Chung, I. and Watts, R. J. (2014). Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J Exp Med 211(2): 233-244.
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  4. Chung, I. (2017). Optical measurement of receptor tyrosine kinase oligomerization on live cells. Biochim Biophys Acta Biomembr 1859 (9 Pt A): 1436-1444.
  5. Chung, I., Akita, R., Vandlen, R., Toomre, D., Schlessinger, J. and Mellman, I. (2010). Spatial control of EGF receptor activation by reversible dimerization on living cells. Nature 464(7289): 783-787.
  6. Chung, I. and Bawendi, M. G. (2004). Relationship between single quantum-dot intermittency and fluorescence intensity decays from collections of dots. Physical Review B 70(16): 165304.
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简介

随着先进的光学成像技术的出现,我们对细胞表面蛋白的调节和功能的认识迅速发展。特别是,利用与抗体结合的明亮荧光团和全内反射荧光显微镜等广域显微镜方法进行单分子追踪(smt)已经成为识别内源性蛋白质如何控制细胞生物学的有价值的工具。然而,一些技术挑战仍然存在;在表面贴装技术中,这些挑战围绕着标记试剂的特性。一个好的试剂应该具有中性(不影响目标蛋白的功能)、标记特异性和明亮的荧光信号。此外,由于与试剂制备相关联的时间和金钱成本,需要较长的保质期。基于半导体的量子点(qdot)或珍妮亚荧光(jf)染料是明亮和可光照的,因此是贴片标记的优秀候选者。中性、高亲和力抗体可选择性结合靶蛋白。然而,抗体的二价性会导致两种蛋白质同时结合,这种桥接效应可以改变蛋白质的功能和行为。利用酶消化中性抗体产生的单价Fab片段可以避免双价性。然而,使用化学交联剂smcc(琥珀酰亚胺4-(n-马来酰亚胺甲基)环己烷-1-羧酸盐)将fab与染料结合需要减少fab片段内的链间二硫键,可降低Fab的结构稳定性,减弱其抗原结合能力。为了解决这个问题,我们使用弱还原剂半胱胺对f(ab’)2进行有限还原,生成fab’片段,半胱胺在铰链区产生游离巯基,而fab’中的链间二硫键是完整的。在这里,我们描述了一种从两种igg亚型中产生高产fab'的方法,并将fab'片段与qdot结合。这种共轭方案可以容易地应用于具有相似化学特性的其他类型的染料。
【背景】细胞表面蛋白功能在其自身环境中受到严格调控。要全面了解它们的功能,就必须监测它们与各种细胞膜成分的相互作用,如其他蛋白质和脂质,以及细胞骨架机械和膜下细胞器。在分子水平上实现基于活细胞成像的光学工具(Joo等人,2008;Lord等人,2010;Chung,2017年)包括常规方法,如共焦显微镜和全内反射荧光显微镜(TIRFM),结合特定模式,如弗斯特共振能量转移(FRET)(Sekar和Periasamy,2003年)、单分子跟踪(SMT)(Moerner,2012年)。荧光相关光谱(fcs)(kim等,2007年)和尖端超分辨率显微镜,如光激活定位显微镜(palm)(betzig等,2006年)、随机光学重建显微镜(storm)(rust等,2006年)。受激发射损耗(STED)显微镜(Hein等人,2008年)、结构光显微镜(Sim)(Gustafsson,2000年和Gustafsson等人,2009年)和晶格光片显微镜(Chen等人,2014年)。使用这些活细胞成像工具来监测内源性蛋白质需要明亮的荧光团偶联试剂,这些试剂与靶蛋白特异性结合。为此,一些明亮的染料如半导体量子点(QDOT)(Dahan等,2003;Chung和Bawendi,2004;Lidke等,2004;Chung等,2010;Bien ly等,2014;Chung和Mellman,2015;Chung等,与高亲和力和非干扰性抗体试剂结合的珍妮亚荧光(JF)染料(Grimm等,2015年和2017年)被广泛使用。然而,抗体可以同时与两种靶蛋白结合。这一问题通常通过使用蛋白质水解酶(如木瓜蛋白酶)消化Fab片段的抗体来解决,木瓜蛋白酶在免疫球蛋白的枢纽区域断裂(Chung等人,2010年;Bien-ly等人,2014年;Chung和Mellman,2015年;Chung等人,2016年)。fab与荧光染料的结合依赖于巯基马来酰亚胺反应。然而,当FAB中的链间二硫键减少时,该反应可使FAB失稳,这导致在相对短的时间内(几周内)对靶蛋白的结合能力丧失。因此,实验室必须经常更新共轭物,造成更高的成本和工时损失。因此,我们使用在铰链区中含有游离巯基的Fab’片段(Selis等<E/EM>2016),它可用于琥珀酰亚胺4 -(N-马来酰亚胺甲基)环己烷-1-羧酸酯(SMCC)的共轭反应,而不降低Fab′中的链间二硫键。为此,我们进行了两步反应,其中igg被胃蛋白酶消化为f(ab')2,fab'由半胱胺有限还原f(ab')2生成。在这个方案中,我们展示了一种从两种不同类型的抗体中产生fab'片段并随后将一种fab'与量子点(qdots)结合以使用smt监测活细胞上egfr的方法。我们相信这种结合方案最有可能提高内源性蛋白质的各种活细胞成像标记试剂的总产量和稳定性。

关键字:单分子示踪, 活细胞成像, 抗体, 量子点, Janelia Fluor (JF)染色, Fab’, Fab, 共轭, 胃蛋白酶

材料和试剂

  1. 吸管头(奥林巴斯,目录号:24-120RL、24-150RL、24-165RL)
  2. 无菌移液管尖端(奥林巴斯,目录号:24-401、24-404、24-412、24-430)
  3. 无菌血清学吸管(奥林巴斯,目录号:12-102、12-104)
  4. 15ml离心管(Olympus,目录号:28-101)
  5. 50毫升离心管(Fisher Scientific,目录号:14-955-239)
  6. 1.5毫升微量离心管(奥林巴斯,目录号:24-281)
  7. 1.5毫升蛋白球管(Eppendorf,目录号:022431081)
  8. 15ml蛋白球锥形管(Eppendorf,目录号:0030122216)
  9. 可调容量吸管(Eppendorf,目录号:2231300008)
  10. Pierce一次性柱(Thermo Scientific,目录号:29920)
  11. NAP-5脱盐塔(GE Healthcare,目录号:17-0853-01)
  12. μ-皿35 mm,高玻璃底(IBIDI,目录号:81158)
  13. 处理过的细胞培养瓶(Thermo Scientific,目录号:12-556-010)
  14. 皮尔斯蛋白浓缩器PES,30K MWCO,0.5毫升(Thermo Scientific,目录号:88502)
  15. 胃蛋白酶(Sigma-Aldrich,目录号:P6887-250mg)
  16. 小鼠igg1同型对照(invitrogen,目录号:02-6100);αegfr(大鼠igg2a)抗体(abcam,目录号:ab231)
  17. mda-mb-468乳腺癌细胞株(atcc,目录号:htb-132)
  18. BCA蛋白质检测试剂盒(Thermo Scientific,目录号:23225)
  19. Superdex G200(GE Healthcare,目录号:17-1043-01)
  20. 磺基SMCC[磺基丁二酰亚胺基4-(n-马来酰亚胺甲基)环己烷-1-羧酸盐],无称重格式(Thermo Scientific,目录号:A39268)
  21. 二甲基亚砜(Thermo Scientific,目录号:20684)
  22. QDOT 605 ITK氨基(PEG)量子点(氨基-PEG-QDOT605,Invitrogen,目录号:Q21501MP)或QDOT 565 ITK氨基(PEG)量子点(氨基-PEG-QDOT565,Invitrogen,目录号:Q21531MP)
  23. HEPES,1 M溶液,pH值7.3,分子生物学级,超纯(Thermo Scientific,目录号:J16924ae)
  24. 氯化钠,5 m(Lonza,目录号:51202)
  25. 半胱胺(Sigma-Aldrich,目录号:M9768-5G)
  26. 染料标记标记,荧光红610 T10(LGC BioSearch Technologies,目录号:RD-5082-5)
  27. 甘油(Thermo Scientific,目录号:J16374AP)
  28. Dulbecco的PBS(Genclone,目录号:25-508)
  29. 胰蛋白酶EDTA(康宁,目录号:25-052-cv)
  30. Pierce F(AB’)2制备工具包(Thermo Scientific,目录号:44988,见注1),包括:
    1. Zeba旋转脱盐塔(Thermo Scientific,目录号:89889)
    2. NAB蛋白A加旋转柱(Thermo Scientific,目录号:89956)
    3. PBS(Thermo Scientific,目录号:1890535)
  31. 醋酸缓冲液,pH 4.0(Fisher Scientific,目录号:50-255-309)
  32. EDTA(费希尔科学公司,目录号:03-500-506)
  33. 细胞生长培养基(RPMI 1640,10%胎牛血清和1%青霉素链霉素)
    1. RPMI 1640,含L-谷氨酰胺,2000 mg/L D-葡萄糖(genclone,目录号:25-506)
    2. 胎牛血清(FBS),热灭活,美国原产地(基因克隆,目录号:25-514H)
    3. 青霉素-链霉素混合物(Lonza,目录号:17-602E)
  34. 醋酸消化缓冲液,pH 4.0(见配方)
  35. 交换缓冲液,pH7.2(见配方)

设备

  1. Eppendorf easypet 3(Eppendorf,目录号:443000018)
  2. pH计(Fisherbrand,Accumet,型号:15)
  3. 涡流混合器(vwr,型号:模拟涡流混合器)
  4. 离心机(Eppendorf,型号:5810R)
  5. 热混合器(Thermo Scientific,型号:13687720)
  6. 端对端混频器(Argos Technologies,Rotoflex,型号:R2000)
  7. CO2培养箱空气套TC(VWR,目录号:10810-902)
  8. 生物安全柜(Labconco,型号:A2)
  9. Nikon Eclipse TE2000倒置显微镜,带TIRF照明器和100X/1.49NA平面APO物镜(Nikon,型号:Eclipse TE2000-E)
  10. ixon背照式EMCCD摄像机(和或技术,目录号:DU-888E-C00-BV-500)
  11. 488nm固体激光器(和或技术)

软件

  1. Java1.80y172的Image J 1.5 2i

程序

这个过程描述了从igg中产生fab'片段及其随后与荧光染料的结合(这里,我们使用qdot),如图1所示。我们使用小鼠igg1优化了消化和还原方案,然后应用这些程序(见注8)进一步优化将αegfr抗体(大鼠igg2a)的fab'片段与qdots结合以进行smt的条件。
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图1。程序概述和卡通示意图。不同抗体之间的反应条件可能不同(见注释5和11)。

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  1. 醋酸消化缓冲液及胃蛋白酶溶液的制备
    1. 制备醋酸盐消化缓冲液(配方1)。
    2. 在醋酸消化缓冲液中制备10 mg/ml胃蛋白酶溶液(见注释2和3)。< >
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  2. igg样品制备(见注4)
    1. 拧下Zeba旋转脱盐塔的底部封盖并松开盖子。将色谱柱置于15毫升离心管中。
    2. 在1000x g下离心柱2分钟以除去存储溶液。弃用直通阀。
    3. 向柱中加入1毫升醋酸消化缓冲液。在1000x g下离心2分钟,并去除通流。重复此步骤三次。
    4. 将平衡柱置于15ml蛋白球锥形管中。取下盖子,缓慢地将0.5毫升抗体样品添加到柱中树脂床的中心。小心不要打扰树脂床。
    5. 更换盖子并在1000x g下离心2分钟。收集igg样品。图2显示,从该缓冲交换步骤中,相对于原始量(车道1),总IgG量略微降低(车道2)。
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  3. 胃蛋白酶消化igg为f(ab’)2
    1. 将0.5ml制备的igg样品移入1.5ml蛋白球锥形管中。
    2. 对于1 mg/ml的igg,向试管中加入2.5μl的10 mg/ml胃蛋白酶溶液。抗体/胃蛋白酶w/w比为20:1。
    3. 让消化反应在37°C的热混合器上进行16小时,以1000转/分的速度搅拌。这种情况可以根据不同的IgG分子而变化(见注释5)。图2中3号车道~100kDa的频带显示f(ab’)2。
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  4. 消化反应终止(见注6)
    1. 拧下Zeba旋转脱盐塔的底部封盖并松开盖子。将色谱柱置于15毫升离心管中。
    2. 在1000x g下离心柱2分钟以除去存储溶液。
    3. 向柱中加入1毫升PBS(pH 7.4)。在1000x g下离心2分钟,并丢弃流过的液体。重复此步骤三次。
    4. 将平衡柱置于15ml蛋白球锥形管中。取下盖子,慢慢地将0.5毫升消化后的IgG样品涂在树脂床的中心。小心不要打扰树脂床。胃蛋白酶消化igg将在这一步终止。
    5. 更换盖子,并在1000x g下离心2分钟以收集流过的液体。参见图2中的车道4。
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  5. f(ab’)2样品的纯化
    1. 让NAB蛋白A+旋转柱和PBS达到室温。将离心机转速设置为1000x g。
    2. 松开NAB蛋白质A+旋转柱上的顶盖,并折断底部封盖。将色谱柱置于15毫升离心管中,离心1分钟以除去存储溶液。
    3. 加入2毫升PBS分散树脂。对于0.5毫升的样品,使用一半的树脂。
    4. 用2毫升PBS平衡柱中的树脂。离心1分钟并丢弃流经的液体。重复此步骤一次。
    5. 用附带的橡胶盖盖住柱底部。将消化液涂在柱上,盖紧顶部。用倒置法对树脂和样品进行再悬浮。在室温下在端对端混合器上培养15分钟。
    6. 松开上盖并拆下下下盖。将色谱柱置于15毫升蛋白球锥形管中,离心1分钟。保留流动(该部分含有F(AB’)2)。参见图2中的5号车道。
    7. 为获得最佳回收率,用0.5毫升PBS清洗色谱柱。离心1分钟,收集流经的液体。重复并收集两个含有额外F(ab’)2的洗涤分数(参见图2中的第6和第7行以及注释7和8)。
    8. 使用bca蛋白质测定法测量蛋白质浓度。
      1. 按照制造商的说明制备BCA试剂和蛋白质标准品的系列稀释液。将200μl BCA试剂与10μl上述标准品或样品混合。
      2. 在37℃下孵育30分钟后,混合物的颜色将根据蛋白质浓度从淡绿色变为紫色。见图3。
      3. 用分光光度计或读板器测量562nm处的吸光度,通过与标准曲线的比较来估计蛋白质浓度。< > < >
        图2。胃蛋白酶消化小鼠IgG1前后的每一步的SDS-PAGE分析。< /强>巷1:最初储存在PBS(pH 7.4)中的整个IgG1样品。通道2:IGG1样品在缓冲液交换后,将其带至醋酸消化缓冲液(pH 4.0)。通道3:在37°C的热振动筛上消化16小时后得到的样品(约100 kDa位置的强带表示F(ab’)2)。通道4:缓冲液交换后的样品,将其带到PBS(pH7.4)以终止反应。通道5:蛋白质A柱在室温下孵育15分钟后的流动。通道6和通道7:从洗涤蛋白质A柱中收集的两个连续部分,以最大化产量。
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        图3。测定纯化后蛋白质浓度的bca法。a.bca溶液的绿紫色转换程度与蛋白质浓度成正比。b.使用吸光度(562nm处)与已知蛋白质浓度(y=0.507x+0.0188)的标准曲线,测定从蛋白质a柱流动中采集的f(ab’)2样品的浓度。
        < >
  6. fab'-qdot共轭物分离柱的制备 注:该片段是Invitrogen对Qdot抗体结合方案的修改。
    1. 通过轻轻摇动或旋涡将分离介质(Superdex G200)悬浮在瓶子中。确保介质完全悬浮,然后开始使用Pierce一次性色谱柱制备色谱柱。
    2. 用两条线标记柱,一条在熔块顶部上方45 mm处,另一条在熔块上方55 mm处(图4a)。
    3. 在装入培养基之前,用纯水湿润熔块。在确保分离介质为均匀悬浮液后,用1毫升移液管将介质装入第二行55毫米标记处(图4b)的柱中,并使其沉降到约45毫米高的填充凝胶床中(图4c)。
    4. 轻轻地在凝胶顶部加入0.5毫升蒸馏水,同时保持床面水平。
    5. 当溶剂水平下降到接近已沉淀凝胶床顶部时,用PBS填充柱,并让PBS水平下降到刚好高于凝胶床顶部。再重复两次。
    6. 当最后一次填充的PBS水平下降到凝胶床顶部上方2至3 mm时,更换底部和顶部盖子。
      < >
      图4。分离柱的制备。a.图像显示熔块上方有两个标记(45和55 mm)。b.凝胶悬浮液填充柱至上部标记(55 mm)。c.凝胶沉淀到下标记处(45 mm)。< >
      < >
  7. 量子点纳米晶的活化(见注9)
    1. 在DMSO中制备新溶解的20 mm磺基SMCC溶液。为此,在229μl二甲基亚砜中溶解2 mg Pierce无称重硫SMCC。
    2. 在氨基-PEG-QDOT605的8μm储备溶液62.5μl中加入1.75μl DMSO中的20 mm磺基SMCC溶液。短暂的漩涡。
    3. 室温下孵育1小时,以500转/分的转速搅拌以激活QDOTS。避光。
    4. 在启动步骤进行时准备脱盐柱。
      < >
  8. 脱盐塔的平衡
    1. 准备交换缓冲液(见配方2)。
    2. 标记NAP-5脱盐塔。一个标记为“减少fab”,另一个标记为“激活qdot”。
    3. 从两个塔上取下顶部和底部盖,让塔中的存储液体排出。当每个柱中的液体接近柱凝胶床的顶部时,开始加入交换缓冲液。
    4. 用10毫升交换缓冲液平衡每一列。
    5. 当在每个柱上的凝胶床上方仍可见交换缓冲液时,将每个柱的底部盖住,并将其放置到F(ab′)2还原和QDOT的活化完成为止。
      < >
  9. fab碎片的产生
    1. 必要时,通过稀释或浓缩,在300μL PBS中制备100μg F(AB’)2。
    2. 当qdot活化几乎完成时,将f(ab’)2样品与半胱胺(5 mm)和edta(2 mm)在10°C下孵育5分钟,以500转/分的速度混合(见注8和注10)。图5第2行的条带代表Fab’(50kDa)和进一步减少的片段(25kDa);后者不太可能保持抗原结合能力。为了最大化Fab’s产率,我们建议使用不同的F(ab′)2个样品,使用小的等分试样(每车道5-10μl)测试各种还原条件(见注释11和12)。 < >
      图5。用半胱胺将F(AB’)2还原成Fab’的SDS-PAGE分析。Lane1:图2中Lane5的F(AB’)2样品。通道2:在pH 7和10°C下,用5毫米半胱胺还原5分钟后的样品(见注11),在缓冲液交换之前。约50 kDa的频带表示fab.
      < >
  10. 还原fab'片段的脱盐和收集
    1. 将40μl蒸馏水加入染料标记物的一小瓶中并混合。这就为两个共轭反应提供了足够的染料标记物。不使用时储存在2-6°C。
    2. 向1.5毫升微量离心管中加入500微升水,并在弯月面处标记管的外侧。再加入500μl水,并在试管外侧做第二个标记,标记的体积为1000μl。扔掉水。该管用于收集步骤j7中的还原fab'和步骤k4中的活化qdot纳米晶。
    3. 当f(ab’)2还原完成(步骤i2)时,向还原fab’中添加20μl染料标记物(在步骤j1中制备)。
    4. 取消标记为“还原Fab”的脱盐柱,并允许剩余的交换缓冲液进入凝胶床。紧接着,将还原的混合物(在步骤J3中制备)添加到凝胶床的顶部。
    5. 允许减少的Fab混合物完全进入凝胶。
    6. 在凝胶床顶部加入1毫升的交换缓冲液洗脱Fab’。
    7. 当第一个有色滴洗脱(见图6)时,开始将还原的Fab'收集到离心管中(在步骤J2中标记);收集不超过500μL(到较低的标记线)。< > < >
      图6。从脱盐柱中收集还原的Fab片段。< /强> a.在还原的Fab′与染料标记的混合物完全进入柱之后,在脱盐柱中加入一毫升交换缓冲液。b.将第一个染色液滴收集到步骤j2中标记的试管中。
      < >
  11. 活化qdot纳米晶的脱盐与收集
    1. 打开标有“活化QDOT”的脱盐塔盖。允许剩余的交换缓冲液进入凝胶床。紧接着,将活化的Qdot nanocrystals(从步骤G3)添加到凝胶床的顶部。
    2. 允许活化的QDOT纳米晶体混合物完全进入凝胶床。
    3. 在凝胶床的顶部加入1毫升的交换缓冲液,将纳米晶包裹起来。
    4. 当第一滴有色物质从柱中洗脱后,开始直接收集到含有还原和脱盐fab'的离心管中。见图7。
    5. 当最终体积达到1毫升时停止收集(达到步骤j2中标记的顶线;500微升活化的量子点纳米晶体)。
    6. 短暂混合。
      < >
      图7。从脱盐柱中收集活化的QDOT。<强> A。在活化Q点完全进入凝胶后,在脱盐柱中加入一毫升交换缓冲液。b.将第一滴染色液收集在试管中。
      < >
  12. 共轭反应
    使还原的fab'和活化的qdot纳米晶在室温下反应2小时。避光。
    < >
  13. 浓缩样品
    1. 将体积(从步骤l1)分成两个蛋白质浓缩器。
    2. 在4000x g下离心15分钟,将每个体积浓缩至~20μl。如果离心后体积为20μl,则继续离心5分钟。
      < >
  14. 量子点共轭fab'与非共轭fab'的分离
    1. 解开分离柱(从步骤F6),并允许剩余的PBS通过重力进入凝胶床。紧接着,将浓缩的共轭反应溶液从两个蛋白质浓缩器(总体积约40μl)中混合到色谱柱顶部。避免干扰凝胶床。
    2. 让共轭反应溶液进入凝胶,然后轻轻加入50μl PBS。让这个缓冲液进入凝胶床。
    3. 用PBS轻轻地填充柱上方的储液罐,让样品通过重力洗脱。目测监控熔块和柱尖之间的“死区”。
    4. 当颜色出现在“死空间”时,在离心管中只收集前8-10滴有色溶液。见图8。
    5. 通过在qdot的第一个吸收峰处的吸光度测量来测定qdot浓度。
    6. 使最终的共轭池达到甘油的50%v/v,并在4°C下储存。
      < >
      图8。从分离柱收集qdot共轭fab。a.共轭混合物完全进入柱后,用pbs填充分离柱。b.当颜色出现在死区时,洗脱液开始收集。
      < >
  15. 用α-egfr-fab'-qdot605结合物在活细胞上追踪egfr
    1. 用胃蛋白酶[20:1抗体/胃蛋白酶(w/w比)]37℃下的醋酸消化缓冲液(pH4.0)搅拌16小时,然后用蛋白质A柱纯化,将α-EGFR抗体消化成F(ab’)2(图9a)。
    2. 用5毫米半胱胺在10℃下将得到的f(ab’)2降低到fab’,持续5分钟(图9b)。
    3. 与活化的qdot605结合产生αegfr fab'-qdot605结合物。
    4. 在全生长培养基中,让2nm的结合物在室温下与镀在玻璃底皿上的活细胞(过度表达egfr的mda-mb-468乳腺癌细胞系)上的靶蛋白(egfr)结合10分钟。用全生长培养基清洗三次(见注13)。
    5. 在100x/1.49na平面载脂蛋白物镜的倒置显微镜上,使用TIRFM在488nm的光激发下进行单分子跟踪(SMT)(我们遵循Chung和Mellman,2015;Chung,2017中描述的方法)。使用EMCCD摄像机在11 Hz处进行图像采集。图9C中显示了使用Image J呈现的单独标记的QDOT的快照图像。
    6. 通过测定与原始抗体结合的表位饱和后结合结合结合物的密度(约为kd的100倍)来估计结合物的结合特异性,相对于未饱和的密度。Fab’-QD结合物的结合特异度为89%。 < >
      图9。用αEGFR抗体(IGG2A)产生SMART的Fab′-QDOT偶联物。< /强> A和B。用αEGFR IgG2a证明Fab’-QDOT605偶联物在SMT上对活细胞的适用性。SDS-PAGE分析α-表皮生长因子受体大鼠IgG2A的胃蛋白酶消化(A)和半胱胺还原(B)。(B)还原产生的IgG的Fab通过量与温度、半胱胺浓度的变化具有相似的相关性。与图5所示小鼠igg1 f(ab’)2的ph值相同(见注11)。c.用αegfr fab'-qdot605结合物标记egfr后,用全内反射荧光显微镜(tirfm)在约60%汇合的mda-mb-468细胞上拍摄一张10.72hz的smt图像(488nm光照,100x油标)。

笔记

  1. 程序A-E修改自Pierce F(AB’)2制备工具包(Thermo Scientific,目录号:44988)的协议。ZEBA自旋脱盐柱,PBS,和NAB蛋白A +自旋柱在此协议中使用,可以在该试剂盒中找到。
  2. 每次反应都应新鲜制备胃蛋白酶溶液。
  3. 胃蛋白酶在高ph缓冲液中不可逆地变性。切勿在中性缓冲液中制备胃蛋白酶溶液。
  4. 当使用浓缩的igg样品时,只需在醋酸盐消化缓冲液中将样品稀释至所需浓度,并跳过步骤b。
  5. 胃蛋白酶消化不同的igg分子。建议在制备f(ab’)2之前,使用小份所需igg进行一个时间过程(1-5和16 h)试验。如果按照程序C中的建议,消化时间设置为16小时,则在一天结束时开始该反应,并在第二天早上停止该反应。
  6. 胃蛋白酶消化后将醋酸缓冲液改为pbs的目的是终止反应,并制备用于蛋白质a纯化(用于去除未消化的igg)的样品,因为蛋白质a柱在酸性ph下无效。
  7. 蛋白A柱可以通过使用IgG洗脱缓冲液(pH 2.8)的制造商说明书再生。
  8. 如图2, 5和9所示,可以根据需要使用非还原性SDS聚丙烯酰胺凝胶电泳来验证消化/还原产物。
  9. 这种共轭方案可以直接应用于马来酰亚胺连接的染料,其中活性马来酰亚胺可以与Fab’中的巯基反应。
  10. 半胱胺对空气和水分敏感。将原瓶保存在真空干燥器中。使用前准备半胱胺溶液。
  11. 半胱胺的还原产率因f(ab’)2样品的不同而不同。强烈建议在pH(5、6和7)、温度(4°C、10°C、22°C和37°C)和半胱胺浓度(0.5-20 mm)的范围内进行中试。
  12. 高效液相色谱法或FPLC可用于进一步纯化Fab′的还原样品。
  13. 结合物的孵育时间和浓度可以根据实验、Fab′、Em>等< 的亲和力而变化,通常在2-10分钟(孵育时间)和0.5~10 nm(共轭浓度)范围内变化。

食谱

  1. 醋酸消化缓冲液,ph 4.0
    0.1 m醋酸缓冲液
    0.01米乙二胺四乙酸
  2. 交换缓冲液,ph值7.2
    50毫米HEPES
    150毫米氯化钠

致谢

我们感谢钟实验室的成员进行了有益的讨论,并感谢jore kotryna vismante拍摄了该过程。这项工作得到了GW癌症中心和KATZEN癌症研究试验奖的支持。从过去的单分子追踪研究(Chung等<E/EM>2010;Bien Ly 等,,2014;Chung 等,,2016)介绍了该方法。

相互竞争的利益

作者声明与此作品无关的财务或非财务竞争利益。

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引用:Teng, I., Bu, X. and Chung, I. (2019). Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells. Bio-protocol 9(18): e3375. DOI: 10.21769/BioProtoc.3375.
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