Analysis of interacting proteins using BIAcore
Analysis of interacting proteins using BIAcore
BIAcore surface plasmon resonance is widely used for (1) studying the interaction process between various biomolecules (e.g., peptides, proteins, oligonucleotides, as well as viruses, bacteria, and small-molecule compounds); (2) specific antibody detection or quality control, disease mechanism, and drug screening; and (3) real-time monitoring of relevant pharmacokinetics, ligand angling, immune modulation, and structure-function relationship.
Operation method
Analysis of interacting proteins using BIAcore
Principle
Biacore is based on Surface Plasmon Resonance (SPR) technology to track interactions between biomolecules in their natural state in real time, without the need for any markers. Surface plasmon resonance (SPR) is an optical phenomenon, in the sensing chip occurs in total reflection interface has a layer of about 50nm thick metal film, polarized light is incident to the prism end, in the prism and the metal film interface will generate surface plasma wave, when the propagation constant of the incident wave and the surface plasmon wave propagation constant match, the free electrons within the metal film will resonate, that is, the surface plasmon resonance, the surface plasmon resonance. When the propagation constant of the incident light wave matches the propagation constant of the surface plasma wave, the free electrons within the metal membrane will resonate, i.e., the surface plasmon resonator. For analysis, a biomolecule, i.e., a ligand (protein, antibody, etc.), is coupled to the surface of the biosensor, and then a solution containing another biomolecule (analyte) that can interact with the target molecule is injected into and flows over the surface of the biosensor. The binding between the biomolecules causes an increase in the surface mass of the biosensor, leading to a change in the refractive index, and the kinetic binding and dissociation constants, affinity, and specificity of the analyte can be automatically obtained by monitoring the angular change in the SPR. Changes in biomolecular interactions are observed.
Materials and Instruments
Protein Samples Move Phase I. Preparation of capture surfaces and binding assays Caveat Sample requirements1. Determine the isoelectric point of the protein to be analyzed by isoelectric focusing or software prediction. Biacore analysis is generally not recommended for proteins with isoelectric points higher than 7 or lower than 3, but special needs can be discussed with the relevant technical staff.Biacore analysis is not recommended for proteins with isoelectric point higher than 7 or less than 3. 2. The target protein (e.g., receptor protein) and the protein to be analyzed should be more than 95% pure when fixed on the sensing slice. 3. The protein solution is dialyzed in PBS (or special buffer for user proteins) and concentrated to a concentration of 200ug/ml to 1mg/ml. 4. Compounds are dissolved in HBS buffer containing 1‰ DMSO and diluted to 10-5M and 10-6M concentrations for preliminary analysis; compounds that are positive in the preliminary analysis are then diluted with buffer containing 1‰ DMSO to a concentration gradient of 10-5M, 5×10-6M, 2×10-6M, 1×10-6M, 5×10-7M and 1×10-7M for The kinetic constants were analyzed. 5. If the compounds cannot be dissolved in HBS buffer containing 1‰ DMSO, the concentration of DMSO can be increased up to 1%. The corresponding concentration of DMSO buffer is prepared as a control. Common Problems This experiment was derived from the next volume of Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell. For more product details, please visit Aladdin Scientific website.
EDC Aminoethanol Extraclean HEPES Buffered Saline Buffer HCl NHS Mouse Anti-TSH Monoclonal Antibody Rabbit Anti-Mouse-Fc Structural Domain TSH TSH Dilution
BIAcore Instruments CM-5 Sensor Chip
Materials
Buffers and solutions
Dilute the storage solution to the appropriate concentration.
EDC [0.2 mol/L aqueous solution of N-ethyl-N'-(dimethylaminopropyl)-carbodiimide]
Aminoethanol (1 ml/L ethanolamine hydrochloride, adjusted to pH 8.5 with NaOH)
Extraclean
Extraclean is a solution for surface regeneration, available from BIAcore.
HEPES buffered saline buffer
10 mmol/L HEPES (pH 7.4)
150 mmol/L NaCl
3 mmol/L EDTA
0.005%(V/V) Tween-20
This buffer can be purchased from BIAcore.
HCl (20 mmol/L)
NHS (0.05 ml/L aqueous N-hydroxysuccinimide)
EDC, aminoethanol and NHS are part of the Amine-coupling Kit purchased from BIAcore.
Antibodies 
Mouse anti-TSH monoclonal antibody (lmg/ml in HEPES buffered saline)
This antibody can be purchased from Alexon Trend, Inc.
Rabbit Anti-Mouse-Fc Structural Domain (RAMc) (30ug/ml in l0 mmol/L pH5.0 sodium acetate solution)
This antibody can be purchased from BIAcore.
TSH (20umol/L in HEPES buffered saline)
TSH can be purchased from Alexon Trend, Inc.
Specialized Devices
BIAcore Instrument
The instrument includes the BIAcore control software and the BIA evaluation software.
Equipment and software can be purchased from BIAcore, Inc. and information is available at www.biacore.com.
CM-5 Sensor Chip
Methods
RAMc by primary amine binding to the surface of the CM-5 sensor chip.
This step takes approx. 45 min. The commands are executed via drop-down menus or icons in the toolbar of the BIAcore control software.
1. Embed the CM-5 SensorChip module into the BLAcore instrument.
2. Filtered and degassed HEPES buffer salt solution is used throughout.
3. 100 tubes containing NHS, EDC, ethanolamine and RAM Fc, and 20 mmol/L HCl, respectively, are placed in the appropriate position in the BIAcore autosampler rack.
4. Place an empty tube in the BIAcore autosampler rack.
This step can be performed if the Immobilization Wizard is used. (Wizards come standard with BlAcore Control Software version 3.0 or later in the BIA2000 and BIA3000.) Using the Wizard's words, add the sample in the specified volume. The target number of RUs combined with RAM Fc is 13000.
5. Start the instrument and flow through a flow cell at a flow rate of 5 ul/min.
6. Add 75ul of NHS to an empty tube.
7. Add another 75ul of EDC to the same tube.
8. Mix the components of the tube containing NHS and EDC.
9. Inject 35ul of NHS/EDC mixture to activate the surface.
10. 35ul of RAM Fc is injected onto the activated surface to bind to the antibody.
11. 35ul of ethanolamine is injected to inactivate excess reactive groups.
12. Rapidly inject 10ul of 20 mmol/LHCl, then remove non-covalently bound material with Extradean.
13. Determine the level of bound RAMc by placing a baseline reporter site prior to the start of RAMc injection and a second reporter site 2 min after the end of the 20 mmol/L HCl injection.
The final RUs of fixed RAMc should be between 10,000 and 13,000; if the resulting value is low, it may be due to the low concentration of RAMc used or to the use of NHS or EDC solutions that have been in storage for more than 2 months. Surfaces with low RUs of RAMc can still be used, but the volume of anti-TSH must be adjusted empirically; typically, more anti-TSH is needed to achieve period binding. TSH to achieve binding levels over time.
14. Turn off the mobile solution, close the command window, and save the report file.
At this point the BIAcore can be placed in a ready (continue) state or the surface can be used directly in the next phase. The RAMc surface is very stable in the machine when the buffer is kept flowing inside the instrument. Alternatively, the chip can be removed and stored in a 50 ml conical tube containing a small amount of water at 4°C; in this way the surface is stable for up to a week.
Detection of anti-TSH binding to the RAMc surface is a step that takes about 20 min. commands are executed via drop-down menus or icons in the toolbar of the BLAcore control software.
15. Start the instrument and set the flow rate of the flow cell containing bound RAMc to 10 ul/min.
16. inject 10ul of 2ug/ml anti-TSH (this requires a total of 40ul of anti-TSH solution).
10ul of anti-TSH will raise approximately 250 KUs. If the RAMc surface does not bind to this level, regenerate (see step 17) and repeat the injection of different volumes of anti-TSH until the desired injection volume is determined.
17. Quickly inject 10ul of 20 mmol/L HCl and regenerate the RAMc surface with Extraclean.
18. To determine the reproducibility of binding to the RAMc surface, repeat the injection of anti-TSH in the volume required to induce 250RUs of binding to anti-TSH.
19. Quickly inject 10ul of 20 mmol/L HCl and regenerate the RAMc surface with Extradean.
20. Shut off the flow-through liquid, close the command window, and save the report file.
Detection of TSH Binding to Captured Anti-TSH Surfaces
This step takes about 20 min. commands are executed via drop-down menus or icons in the toolbar of the BIAcore Control Software.
21. Start the instrument and set the flow cell containing bound RAMc to a flow rate of 10 pl/min.
22. Inject 10ul of 2ug/ml anti-TSH (or the volume required to induce 250RUs of anti-TSH binding as measured in steps 15-20).
23. inject 25ul of 200nmol/L TSH, limiting the dissociation time to 120s (this requires a total of 65ul of TSH solution). 120s of dissociation time is sufficient to measure the rate of dissociation of the antigen-antibody complex.
After injection of approximately 20ul of 200nmol/L TSH, the curve flattens (the slope reaches zero), indicating a transient steady state in the interaction between the antigen and antibody components. If the curve does not flatten, regenerate the surface and repeat steps 21-23 after changing the TSH concentration to 400 nmol/L in step 23 (see Figure 18-26 and Figure 18-27).
24. Quickly inject 10ul of 20 mmol/LHCl, then regenerate the RAMc surface with Extraclean.
25. Turn off the flow stream, close the command window, and save the report file. 
Phase II Kinetic Analysis of Antigen-Antibody Interactions
MATERIALS
Buffers and solutions
Dilute the storage solution to the appropriate concentration.
HEPES buffered saline buffer or appropriate rinse buffer
10 mmol/LHEPES (pH 7.4)
150 mmol/LNaCl
3 mmol/LEDTA
0.005%(V/V) Tween-20
This buffer can be purchased from BIAcore.
Antibodies
Mouse anti-TSH monoclonal antibody (lmg/ml, in HEPES buffer salt solution)
This antibody can be purchased from Alexon Trend, Inc.
Dilute the antibody to 2ug/ml and prepare 1000ul or the desired volume) to achieve the target resonance units measured in Steps 15-20 of Phase I. The antibody should be diluted to 2ug/ml and prepared in 1000ul or the desired volume.
TSH (20umol/L, dissolved in HEPES buffered salt solution)
TSH can be purchased from Alexon Trend, Inc.
Prepare a series of TSH dilutions for injection:
1. Different dilutions of 200, 100, 50, 25, 10, and 5 nmol/L were prepared and each was dissolved in 280ul of flow buffer for experiments.
2. Dispense 70ul of each concentration into four 7 mm plastic vials and cap each vial.
3. Place the vials in the appropriate position in the BIAcore rack as indicated by the sample cycling parameters.
Specialized Devices
BIAcore Instrument
The instrument includes the BIAcore control software and the BIA evaluation software.
Equipment and software can be purchased from BIAcore, Inc. and information is available at www.biacore.com.
CM-5 sensing chip, combined with RAMc as described in Phase 1.
METHODOLOGY
The following program consists of three method modules or segments: the Main Program, the Define Analysis Program (DEFINEAPROG), and the Define Sample. Note that there are four DEFINEAPROG modules, each of which represents a section of the main program (Module 1, Module 2, Module 3, and Module 4). When editing a method with the software, method commands should be written in upper case letters and user-defined parameters should be written in lower case letters.
Partial comments followed by an exclamation point (!) followed by an exclamation point (!) are considered text rather than command code and are inserted to explain the meaning of the previous command. 
