Protein Interaction Analysis Using an IASYS Biosensor 【Kitto Lab, The Universit

Immobilization of ligands on cuvette surfaces and measure the interactions of ligand which is immobilized on the cuvette and the ligate which is added to the immobilized cuvette.

Immobilization of ligands to the cuvette.

Immobilization of ligands to the carboxymethylated dextran coated cuvette

1. Dissolve 0.2 g N-Hydroxysuccinimide (NHS) in 15 ml of deionized H2O to get 0.0133 g/ml (0.116 mol/L) concentration and aliquot into 250 µl volume and store in a -20 °C freezer.
2. Dissolve 1.15 g 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in 15 ml of deionized H2O to get 0.077 g/ml (0.400 mol/L) concentration and aliquot into 250 µL volume and store in a -20 °C freezer.
3. Thaw one vial of each NHS and EDC
4. Incubate the cuvette to thermally equilibrate by pipetting 200 µl of PBS/T buffer (10 mM PBST 0.05% Tween-20) and wait until a stable baseline is obtained ( the changes of the signal response were smaller than 3 arc seconds in 10 minutes) and obtain a PBS/T buffer base line for 7 minutes.
5. Immediately prior to use, thoroughly mix equal volumes of NHS and EDC solutions. Add 200 µl of the mixture to the cuvette while sip up the PBS/T buffer and activate the cuvette for 7 minutes.
6. Remove unreacted activation mixture and wash with PBS/T buffer for 2 minutes.
7. Add ligate solution at optimized concentration and pH for 10 minutes.
8. Remove non-coupled ligand with a PBS/T buffer and wash for 2 minutes. If the arcsec of the immobilized ligand is less than expected, repeat step 7 and 8.
9. Add 1 M ethanolamine, pH 8.5, for 2 minutes to block the activated site of the cuvette.
10. Wash with PBS/T buffer for 5 minutes and calculate the amount of immobilized ligand.

Immobilization of ligands to aminosilane cuvette

All solutions for immobilization were prepared in 10 mM sodium phosphate buffer (10 mM, pH 7.7) which was made with ultrapure water and filtered by 0.2 mm polyethersulfone sterilized membrane.

1. Add 200 µl of sodium phosphate buffer (10 mM, pH 7.7) to the cuvette and obtain a stable baseline (the changes of the signal response were smaller than 3 arc seconds in 10 minutes).
2. Aspirate and sodium phosphate buffer. Add 200 µl of BS3 solution (1mM, 0.57 mg/ml) and wait for 15 minutes.
3. Aspirate the BS3 and add 200 µl sodium phosphate buffer to wash the cuvette three times.
4. Once a stable baseline was achieved, Add ligate solution at optimized concentration and wait for 20 minutes.
5. Wash the cuvette three times with 200 µl sodium phosphate buffer. The difference in the pre- and post-immobilization responses was used to calculate the amount of ligand immobilized.
6. Block any remaining sites on the activated surface of the cuvette by addition of 200 µl of 2 mg/ml bovine serum albumin for 5 minutes.
7. Aspirate the cuvette and wash three times with 10 mM sodium phosphate buffer and the sodium phosphate buffer was then changed to PBST buffer (10 mM phosphate-buffer saline, 0.05% v/v Tween-20).

Interaction analysis

After the ligand was immobilized to the hydrogel surface of the cuvette, the IAsys system was used to test a series of different samples or different concentrations of the same sample.

Each binding cycle was performed with constant instrument parameters (temperature: 23 °C, stirring speed: 50 revolutions/minute, sampling interval: 2 seconds, smoothing: 5).

1. Establish a PBST buffer baseline for 5 minutes.
2. Add the ligate by spiking the PBST buffer with calculated volumes of the stock ligate solution. The association interaction was monitored for 9 minutes.
3. Wash the cuvette with 10 mM PBST for 3 minutes to remove the unbound antibody.
4. Add the regenerate solution to the cuvette for 2 minutes to removed the ligate and regenerate the immobilized ligand.
   

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