Difference between revisions of "20.109(S18):Confirm ligand binding using differential scanning fluorimetry assay (Day6)"
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#Perform the necessary calculations to complete the above chart for a total reaction volume of 10 μL. | #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. | #*Confirm your values with the teaching faculty before proceeding. | ||
| − | #Each team will setup triplicate reactions for | + | #Each team will setup triplicate reactions for 7 different conditions: |
| − | #*Condition 1: control protein ( | + | #*Condition 1: control protein (assay control) |
| − | #*Condition 2: control protein AND control substrate ( | + | #*Condition 2: control protein AND control substrate (assay control) |
| − | #*Condition 3: no protein | + | #*Condition 3: no protein (internal control) |
| − | #*Condition 4: FKBP12 | + | #*Condition 4: FKBP12 AND DMSO (internal control) |
#*Condition 5: FKBP12 AND rapamycin | #*Condition 5: FKBP12 AND rapamycin | ||
#*Condition 6: FKBP12 AND ligand #1 | #*Condition 6: FKBP12 AND ligand #1 | ||
#*Condition 7: FKBP12 AND ligand #2 | #*Condition 7: FKBP12 AND ligand #2 | ||
| − | #Generate a chart, or list, that details what reagents will be in each master mix for | + | #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. | #*All reactions will contain dye. | ||
#*Only reactions without rapamycin or ligand will contain DMSO. | #*Only reactions without rapamycin or ligand will contain DMSO. | ||
| Line 62: | Line 62: | ||
#Use the values calculated in Step #3 to prepare your master mixes in labeled 1.5 mL centrifuge tubes. | #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. | #*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. | ||
| + | #Next, prepare the master mixes for your assay controls, Conditions #1 and #2. | ||
| + | #* | ||
#When you have prepared your master mixes, take them to the front laboratory bench. | #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=== | ===Part 3: Examine binding shifts=== | ||
Revision as of 16:44, 9 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 | |
| PBS (1X) | add for a total of 10 μL reaction | dependent upon master mix |
- 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 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
- 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.
- Obtain the appropriate aliquots from the front laboratory bench.
- 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.
- Next, prepare the master mixes for your assay controls, Conditions #1 and #2.
- 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
Next day: Complete data analysis
