Difference between revisions of "20.109(S18):Confirm ligand binding using differential scanning fluorimetry assay (Day6)"

Introduction

Interactions between low molecular weight ligands and proteins can been shown to increase the thermostability of proteins. This means that proteins bound to ligand are able to maintain tertiary structure, or resist denaturation, at higher temperatures than unbound proteins. Today we will use differential scanning fluorimetry (DSF) to examine the potential FKBP12 binders identified in our SMM screen.

DSF is a method used to identify low molecular weight ligands that bind and stabilize a protein of interest. In this assay, protein denaturation is measured via a fluorescent dye that has an affinity to hydrophobic regions. When the protein is folded the hydrophobic pockets are inaccessible to the dye and the fluorescent signal is quenched by water in the solution. As the protein unfolds, the dye interacts with the hydrophobic regions and emits a fluorescent signal that can be detected.

When a protein is bound to a ligand, the stability can be increased such that the temperature at which the protein denatures is increased. In the DSF assay, this is measured as a shift in the T_m, or melting temperature, which is defined as the temperature at which 50% of the protein is unfolded. This value represents the midpoint of the transition from structured (folded) to denatured (unfolded).

The ΔT_m is the difference between the T_m of the unbound protein sample, or protein sample without added ligand, and the bound protein sample, protein sample with added ligand. If the tested ligand binds the protein of interest, the ΔT_m can be observed as a shift in the plotted DSF data. For example, the data below show results of a pilot experiment completed in preparation for this module. The…

Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on writing impactful abstracts and titles.

Part 2: Prepare samples for DSF assay

As in the previous laboratory session, you will prepare master mixes for the conditions you will test. Because the master mixes for the DSF assay are more complicated, the below chart will assist you in completing the required calculations.

1. Perform the necessary calculations to complete the above chart for a total reaction volume of 10 μL.
   • Confirm your values with the teaching faculty before proceeding.
2. Each team will setup triplicate reactions for 7 different conditions:
   • Condition 1: control protein (assay control)
   • Condition 2: control protein AND control substrate (assay control)
   • Condition 3: no protein (internal control)
   • Condition 4: FKBP12 AND DMSO (internal control)
   • Condition 5: FKBP12 AND rapamycin
   • Condition 6: FKBP12 AND ligand #1
   • Condition 7: FKBP12 AND ligand #2
3. Generate a chart, or list, that details what reagents will be in each master mix for Conditions #3 - #7 listed above.
   • All reactions will contain dye.
   • Only reactions without rapamycin or ligand will contain DMSO.
   • Include the volume of each reagent (for a final volume of 3.25 the reaction volume, which is 10 μL) as each condition will be tested in triplicate.
   • Again, confirm your values with the teaching faculty before proceeding.
4. Obtain the appropriate aliquots from the front laboratory bench.
5. Use the values calculated in Step #3 to prepare your master mixes in labeled 1.5 mL centrifuge tubes.
   • You will add all reagents except FKBP12 protein, as the teaching faculty will add the protein to the samples immediately prior to measuring the fluorescence signal.
6. Next, prepare the master mixes for your assay controls, Conditions #1 and #2.
7. When you have prepared your master mixes, take them to the front laboratory bench.
   • Be sure that all tubes are clearly labeled!

Part 3: Examine binding shifts

Reagents

• FKBP12, Abcam
• DSF dye, Thermo Fischer
• ligands, Chembridge

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