Difference between revisions of "20.109(S18):Confirm ligand binding using differential scanning fluorimetry assay (Day6)"
From Course Wiki
Noreen Lyell (Talk | contribs) (→Part 2: Prepare samples for DSF assay) |
Noreen Lyell (Talk | contribs) (→Part 2: Prepare samples for DSF assay) |
||
| Line 38: | Line 38: | ||
| | | | ||
|- | |- | ||
| + | | PBS (1X) | ||
| + | | add for a total of 10 &mu:L reaction | ||
| + | | dependent upon master mix | ||
|} | |} | ||
</center> | </center> | ||
| Line 43: | Line 46: | ||
#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 5 different conditions: | + | #Each team will setup triplicate reactions for 5 (?) different conditions: |
#*Condition 1: control protein (?) | #*Condition 1: control protein (?) | ||
#*Condition 2: control protein AND control substrate (?) | #*Condition 2: control protein AND control substrate (?) | ||
| Line 51: | Line 54: | ||
#*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 the the conditions | + | #Generate a chart (or list) that details what reagents will be in each master mix for the the conditions listed above. |
| − | #*Include the volume of each reagent. | + | #*Include the volume of each reagent (for a final volume of 3.25 the reaction volume). |
| + | #*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, | ||
===Part 3: Examine binding shifts=== | ===Part 3: Examine binding shifts=== | ||
Revision as of 22:39, 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 | |
| PBS (1X) | add for a total of 10 &mu:L 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 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 listed above.
- Include the volume of each reagent (for a final volume of 3.25 the reaction volume).
- 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,
Part 3: Examine binding shifts
Reagents
- FKBP12, Abcam
- DSF dye, Thermo Fischer
- ligands, Chembridge
Next day: Complete data analysis
