The SPR measurements were performed using BIAcore S200 (Cytiva, Marlborough, USA) following a protocol provided by the manufacturer. First, the carboxymethyl dextran matrix on CM5 sensor chip (Cytiva) was activated by injection of a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Recombinant proteins in 10 mmol/L acetate buffer (pH 4.5, GE Healthcare) was then injected over the chip surface at a flow rate of 30 μL/min to couple the amino groups of the recombinant proteins to the carboxymethyl dextran matrix. After the coupling reaction, the remaining activated ester groups were deactivated by ethanolamine. The binding study was carried out at 25℃ in phosphate buffer saline (PBS, 20 mmol/L phosphate buffer with 2.7 mmol/L KCl, 137 mmol/L NaCl, and 0.05% Tween-20, pH 7.4). The heparin molecule or fondaparinux at different concentrations were flowed over the immobilized recombinant proteins at a flow rate of 30 μL/min with a contact time of 60 s and a dissociation time of 100 s. The surface was regenerated by injection of 10 mmol/L glycine-HCl (pH 2.5) at a flow rate of 30 μL/min for 30 s. Data was collected and analyzed using the BIA evaluation software (Version 1.0, Cytiva).
Acetate buffer
Manufactured by GE Healthcare
Acetate buffer is a chemical solution used to maintain a specific pH range in laboratory applications. It is a common buffer solution employed in various analytical and experimental procedures, such as electrophoresis, enzyme assays, and cell culture. The acetate buffer helps to stabilize the pH of the system, ensuring that the desired chemical reactions or biological processes can occur optimally.
Automatically generated - may contain errors
Lab products found in correlation
Hbs ep buffer, by GE Healthcare (2 mentions)
Cm5 sensor chip, by Cytiva (1 mentions)
Biacore s200, by Cytiva (1 mentions)
Ethanolamine, by GE Healthcare (1 mentions)
Cm5 sensor chip, by GE Healthcare (1 mentions)
Biacore t100, by GE Healthcare (1 mentions)
Vitronectin, by R&D Systems (1 mentions)
Cm7 chip, by GE Healthcare (1 mentions)
Fibronectin, by Roche (1 mentions)
Recombinant human integrin αvβ3, by R&D Systems (1 mentions)
4 protocols using acetate buffer
1
Surface Plasmon Resonance Binding Analysis
2
Optical Biosensor-Based Carbonic Anhydrase Assay
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Experimental data sets were generated with a BIACORE T100 optical biosensor equipped with research-grade CM5 sensor chips (GE Healthcare, Baie d’Urfe, QC). HBS-EP buffer, acetate buffer and ethanolamine were purchased from GE Healthcare. N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), carbonic anhydrase isozyme II (CAII) that had been purified from bovine erythrocytes, 4-carboxybenzenesulfonamide (CBS), sulfanilamide, 1,3-benzenedisulfonamide (BDS), Sulpiride, Furosemide, 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), dimethyl sulfoxide (DMSO) and phosphate buffer saline (PBS, 10 mM, pH 7.4) were purchased from Sigma-Aldrich Canada Ltd (Oakville, ON).
3
IGF-1R Binding Kinetics Analysis
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The Biacore T200 instrument was prepared by washing the CM5 chip with 50 mM of NaOH. All flow cells were activated for amine coupling with EDC/NHS, after which reference flow cells were blocked with ethanolamine, and sample cells were loaded with 5.5 μg/mL of IGF-1R protein in 10 mM of acetate buffer (Cat No: BR-1003-49, GE Healthcare).
Samples of MYL-1501D and reference products were diluted in HBS-EP buffer (Cat No: BR-1001-88, GE Healthcare) to concentrations of 3.00, 1.50, 0.75, 0.38 (run-in duplicate), 0.19, and 0.09 μM. Running buffer was processed through the instrument for 5 start-up cycles, followed by addition of the diluted samples in buffer at a flow rate of 50 μL/min maintained for 60 seconds for association before dissociation with buffer alone for 150 seconds. Kinetic run data traces were evaluated using a 1:1 Langmuir model with RI set to 0.
Samples of MYL-1501D and reference products were diluted in HBS-EP buffer (Cat No: BR-1001-88, GE Healthcare) to concentrations of 3.00, 1.50, 0.75, 0.38 (run-in duplicate), 0.19, and 0.09 μM. Running buffer was processed through the instrument for 5 start-up cycles, followed by addition of the diluted samples in buffer at a flow rate of 50 μL/min maintained for 60 seconds for association before dissociation with buffer alone for 150 seconds. Kinetic run data traces were evaluated using a 1:1 Langmuir model with RI set to 0.
4
Binding of 10Fn3 Molecules to Integrin
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Example 13
Recombinant human integrin αVβ3 (R&D Systems, Minneapolis Minn.) was diluted to 40 ug/mL in Acetate buffer pH 5.0 (GE Healthcare, Piscataway N.J.), and. then immobilized on a CM7 chip (GE Healthcare) using standard amine coupling techniques. 500 nM fibronectin (Roche Diagnostics, Indianapolis, Ind.) and vitronectin (R&D Systems) and 5 μM of either non-binding control 10Fn3 molecule (consisting of SEQ ID NO: 6 with an additional MG at the N-terminus and with a single amino acid substitution that changes RGD to RGE) or targeted 10Fn3 molecules (having a mutated FG loop that does not contain an RGD motif) were flowed over the top of the immobilized integrin. Binding RU was collected at the end of the sample injection. The results indicate that the lack of RGD in the FG loop results in abolishing binding of 10Fn3 molecules to fibronectin and vitronectin.
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