Difference between revisions of "20.109(S18):Confirm ligand binding using differential scanning fluorimetry assay (Day6)"
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===Part 2: Prepare samples for DSF assay=== | ===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. | |
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| + | #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. | ||
| + | #Each team will setup triplicate reactions for 5 different conditions: | ||
| + | #*Condition 1: control protein (?) | ||
| + | #*Condition 2: control protein AND control substrate (?) | ||
| + | #*Condition 3: no protein | ||
| + | #*Condition 4: FKBP12 | ||
| + | #*Condition 5: FKBP12 AND rapamycin | ||
| + | #*Condition 6: FKBP12 AND ligand #1 | ||
| + | #*Condition 7: FKBP12 AND ligand #2 | ||
| + | #Generate a chart (or list) that details what reagents will be in each master mix for the the conditions to be tested. | ||
| + | #*Include the volume of each reagent. | ||
===Part 3: Examine binding shifts=== | ===Part 3: Examine binding shifts=== | ||
Revision as of 22:32, 3 February 2018
Contents
Introduction
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.
| Reagent (stock concentration) | Final concentration of stock reagent in reaction | Volume of stock reagent in reaction |
|---|---|---|
| FKBP12 (1 mg/mL) | 1.2 μg/mL | |
| DMSO (1%) | 0.2% | |
| rapamycin (50 μM) | 10 μM | |
| ligand (200 μM) | 40 μM | |
| dye (5X) | 1X |
- 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.
- Each team will setup triplicate reactions for 5 different conditions:
- Condition 1: control protein (?)
- Condition 2: control protein AND control substrate (?)
- Condition 3: no protein
- Condition 4: FKBP12
- Condition 5: FKBP12 AND rapamycin
- Condition 6: FKBP12 AND ligand #1
- Condition 7: FKBP12 AND ligand #2
- Generate a chart (or list) that details what reagents will be in each master mix for the the conditions to be tested.
- Include the volume of each reagent.
Part 3: Examine binding shifts
Reagents
- FKBP12, Abcam
- DSF dye, Thermo Fischer
- ligands, Chembridge
Next day: Complete data analysis
