Difference between revisions of "20.109(F21):M2D5"

20.109(F21): Laboratory Fundamentals of Biological Engineering

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       Module 1: Genomic instability                          Module 2: Drug discovery       

Introduction

describe DSF...

Protocols

A. Protein biotinylation for immobilization on sensor probe. The protein needs to be biotinylated to facilitate immobilization to the Streptavidin BLI probe. A detailed protocol for how this is done is available here. For these experiments, you will be provided with a biotinylated protein stock that is at 0.5 µM in 1x PBS, pH 7.4, ## mM TCEP (disulfide reducing agent).

B. Assay plate setup. Two teams will share an assay plate and run samples together on the Octet BLI instrument. Five dilutions of the test compound will be evaluated. a. Each team is responsible for preparing the set of samples described in the <plate map> for that team. You will prepare samples in Eppendorf tubes, and transfer them to the specified well positions on the shared 96-well assay plate. b. Arrange Eppendorf tubes in a row in a rack and label (left to right) as outlined below: i. B = Buffer (1x PBS, pH 7.4 + 1mM TCEP) ii. L = Protein “loading” solution iii. N = Neutralization (biocytin) solution iv. S1 = 2.5 µM test compound (Sample 1) v. S2 = 5 µM test compound (Sample 2) vi. S3 = 10 µM test compound (Sample 3) vii. S4 = 20 µM test compound (Sample 4) viii. S5 = 40 µM test compound (Sample 5) c. Add 500 µL of buffer, protein and biocytin solutions to the Eppendorf tubes labeled B, L and N, respectively. d. Prepare a 10 µM working stock of test compound in tube S5: i. Add 1 mL of 1x PBS, pH 7.4 to tube S5. ii. Add 4 µL of 10 mM compound stock to tube S5. iii. Vortex to mix thoroughly. e. Prepare serial 2-fold dilutions of test compound in Tubes S1 – S4 as follows: i. Add 500 µL of 1x PBS, pH 7.4 to each of tubes S1 – S4. ii. Tube S4: Add 500 µL of solution from Tube S5 to Tube S4, and vortex thoroughly to mix. iii. Tube S3: Add 500 µL of solution from Tube S4 to Tube S3, and vortex thoroughly to mix. iv. Tube S2: Add 500 µL of solution from Tube S3 to Tube S2, and vortex thoroughly to mix. v. Tube S1: Add 500 µL of solution from Tube S2 to Tube S1, and vortex thoroughly to mix. f. Transfer 200 µL of each solution from Eppendorf tubes B, L, N, S1-S5 to the wells assigned to your team’s assay plate according to the plate map. NOTE. In Column 2, you will add solution L (protein loading solution) to the top row. However, in the row below, you will add solution B (buffer). This design allows you to have a “reference” probe so you can observe how the test compound interacts with the probe, and subtract out any contribution this makes to the signal observed from the protein-loaded probe. g. Cover the plate until it is your turn to analyze it on the Octet instrument in the Biophysical Instrumentation Facility (BIF) located in 68-470.

A. Running samples on Octet BLI instrument. As you’ve seen in the video, the Octet automates the introduction of multiple probes into samples, and these can be moved in parallel (to the left or right) to achieve the sequence of events needed for performing quantitative binding and dissociation assays. The following pre-set sequence will be used in your assays. a. Probes immersed in Column 1 (Buffer) to begin before moving to Column 2. In Column 2, protein “Loading” onto the probes will occur in odd-numbered rows, while no protein will be loaded onto the reference probes in even-numbered rows. i. You should observe an increase in binding signal as protein binding to the probe occurs, while signal from the probe immersed in buffer remains flat. b. Probes moved back to Column 1 (“Wash” step). [Note: Biotin binds streptavidin with very high affinity, and no appreciable dissociation of the protein will be observed during this step]. c. Probes moved to Column 3 containing biocytin to block all unoccupied biotin binding sites on the protein and reference probes (“Neutralization” step). This should eliminate additional signal (background) due to test compound binding to unoccupied biotin binding sites on streptavidin. d. Probes moved back to Column 1 (“Wash” step). e. Probes moved to Column 4 (“Association” step for the lowest test compound concentration, S1). i. Data collected on the protein-loaded probe reflects compound “associating” with the protein to form a protein-small molecule complex. ii. Ideally, little association of compound to the probe without protein should occur. However, as the test compound concentration is increased (i.e., from S1-S5), increased compound binding to the naked probe may occur. However, this should be lower in magnitude than what is observed for the protein-loaded probe. f. Probes moved to Column 1 (“Dissociation” step). i. In this step, the small molecule unbinds from the protein complex on the probe, and gets (infinitely) diluted in the bulk buffer. The signal should exponentially decay. g. Steps (e) and (f) above are repeated for Columns 5-8 to obtain a series of association and dissociation data at progressively increasing concentrations of test compound.

Reagent list

• DSF dye (Thermo Fisher)
• ligands (Chembridge)

Navigation links

Next day: Perform secondary assay to test putative small molecule binders