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

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==Introduction==
 
==Introduction==
  
describe DSF...
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To test the 'hits' that were identified in the SMM screen, you will perform a BioLayer Interferometry (BLI) experiment. The BLI assay is used to measure biomolecular interactions by assessing the interference pattern of white light. In this assay, a change in the number of molecules bound to a biosensor tip result in a shift in the interference pattern. These shifts can be measured in real-time.
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To measure biomolecular interactions, a layer of the protein of interest is immobilized on a biosensor tip.  When the immobilized protein binds to analyte in a solution an increase in the optical thickness at the biosensor tip results in a wavelength shift.  The shift is a direct measure of the change in thickness of the biological layer.  Because the shift is caused by thickness of the biological layer at the biosensor tip, only the binding or dissociating of analyte generates an interference pattern.
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[[Image:Fa21 M2D5 BLI kinetics.png|thumb|center|500px|'''Kinetics of binding in BLI assay.''' Image from https://2bind.com/bli/]]
  
 
==Protocols==
 
==Protocols==

Revision as of 05:25, 30 October 2021

20.109(F21): Laboratory Fundamentals of Biological Engineering
Drawing provided by Marissa A., 20.109 student in Sp21 term.  Schematic generated using BioRender.

Fall 2021 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       Module 1: Genomic instability                          Module 2: Drug discovery       


Introduction

To test the 'hits' that were identified in the SMM screen, you will perform a BioLayer Interferometry (BLI) experiment. The BLI assay is used to measure biomolecular interactions by assessing the interference pattern of white light. In this assay, a change in the number of molecules bound to a biosensor tip result in a shift in the interference pattern. These shifts can be measured in real-time.

To measure biomolecular interactions, a layer of the protein of interest is immobilized on a biosensor tip. When the immobilized protein binds to analyte in a solution an increase in the optical thickness at the biosensor tip results in a wavelength shift. The shift is a direct measure of the change in thickness of the biological layer. Because the shift is caused by thickness of the biological layer at the biosensor tip, only the binding or dissociating of analyte generates an interference pattern.

Kinetics of binding in BLI assay. Image from https://2bind.com/bli/

Protocols

Part 1: Prepare biotinylated PF3D7_20109-F21

Protein biotinylation is necessary for immobilization on a sensor probe. Specifically, the PF3D7_20109-F21 protein needs to be biotinylated to facilitate immobilization to the Streptavidin BLI probe. For timing reasons, this step was completed in the Niles Laboratory. A detailed protocol for how this was done is linked here for you to review during any downtime in class today.

For your 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).

Part 2: Use biotinylated PF3D7_20109-F21 in BLI assay

You will prepare the samples for the BLI assay in 96 well plates. Two teams will share an assay plate and run samples together on the Octet BLI instrument. In your experiments, five dilutions of the test compound will be evaluated.

  1. Each team is responsible for preparing the set of samples according to the plate map for your team! Your Instructor will provide a handout with the specific plate map that should be used to each team. A generic plate map is provided below for reference.
    Fa21 M2D5 generic plate map.png
  2. You will prepare samples using eight Eppendorf tubes, and transfer them to the specified well positions on the shared 96-well assay plate according to your plate map.
  3. Arrange and label Eppendorf tubes in a row in a rack and label (left to right) as outlined below:
    • B = Buffer (1x PBS, pH 7.4 + 1mM TCEP)
    • L = Protein “loading” solution
    • N = Neutralization (biocytin) solution
    • S1 = 2.5 µM test compound (Sample 1)
    • S2 = 5 µM test compound (Sample 2)
    • S3 = 10 µM test compound (Sample 3)
    • S4 = 20 µM test compound (Sample 4)
    • S5 = 40 µM test compound (Sample 5)
  4. Add 500 µL of buffer, protein and biocytin solutions to the Eppendorf tubes labeled B, L and N, respectively.
  5. Prepare a 10 µM working stock of test compound in tube S5:
    • Add 1 mL of 1x PBS, pH 7.4 to tube S5.
    • Add 4 µL of 10 mM compound stock to tube S5.
    • Vortex to mix thoroughly.
  6. Prepare serial 2-fold dilutions of test compound in Tubes S1 – S4 as follows:
    • Add 500 µL of 1x PBS, pH 7.4 to each of tubes S1 – S4.
    • Tube S4: Add 500 µL of solution from Tube S5 to Tube S4, and vortex thoroughly to mix.
    • Tube S3: Add 500 µL of solution from Tube S4 to Tube S3, and vortex thoroughly to mix.
    • Tube S2: Add 500 µL of solution from Tube S3 to Tube S2, and vortex thoroughly to mix.
    • Tube S1: Add 500 µL of solution from Tube S2 to Tube S1, and vortex thoroughly to mix.
    • 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.
    • IMPORTANT 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.
  7. 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.

Part 3: Perform BLI experiment

You will run your samples on the 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.

  1. 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.
    • 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.
  2. 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].
  3. 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.
  4. Probes moved back to Column 1 (“Wash” step).
  5. Probes moved to Column 4 (“Association” step for the lowest test compound concentration, S1).
    • Data collected on the protein-loaded probe reflects compound “associating” with the protein to form a protein-small molecule complex.
    • 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.
  6. Probes moved to Column 1 (“Dissociation” step).
    • 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.
  7. Steps #5 and #6 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

  • Biotinylation reagent (NHS-PEG4-Biotin) (from ThermoFisher Scientific)
  • Biocytin (1 µg/mL)
  • Biotinylated PF3D7_20109-F21 protein (0.5 µM)
  • Buffer (1x PBS, pH 7.4)
  • Streptavidin probes (from Sartorius)
  • Compounds (10 mM stocks in DMSO)
  • Biolayer Interferometry (BLI) instrument (Octet-Red)

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