Protocol 021 - TBXT SPR compound screen
Introduction
TBXT compound profiling must be performed using freshly purified brachyury or immediately-thawed protein that was flash-frozen and stored at -80C.
Materials
Procedure
SPR Running Buffer: Prepare 1L without DMSO. Pass through a 0.2 um filter. Remove 20 mL of running buffer and store in 50 mL conical, and add 20 mL DMSO -- 2% final concentration.
Wash Buffer #1: Prepare 25 mL and pass through a 0.2 um filter.
Wash Buffer #3: Prepare 25 mL and pass through a 0.2 um filter
Prepare Solvent Correction stocks and 4-point solvent correction working solutions
In order to reach proper Running Buffer volume, add 10 mLs to 15 mL conical and then pipet out whatever volume of DMSO is needed
Move Line A to SPR Running Buffer
Set Flow Cell Temperature to 25C
Set Sample Compartment Temperature to 25C
Dock SA-Chip (Note Lot/Serial Number)
Change Solution (~ 6 min)
Dilute pure TBXT with SPR Running Buffer
Pass pure TBXT through centrifugal filter, 10000 g for 1 min
Go to Methods Tab -- Empy Method -- Empty Immobilization Method
Add Step - SA biotin capture
Go to Position and Plate Layout
- Immobilize on a select number of channels (1-8)
- There are eight channels but you do not need to use all 8
Set Immobilization to 480 sec, 5 uL/min
Load plate according to plate layout sheet
You should achive > 7,000 Response Units (RU)
Theoretical Rmax Calculator
Dilute compounds in 100% DMSO to 200 uM final concentration (0.5% final DMSO concentration)
- Make 200 uM stock in 250 uL SPR running buffer
- Heat to 40 or 50C with sonication for 5-10 min as needed
- This should be performed prior to going to the facility
Select Binding Screen - LMW
- Set binding to 60 sec at 10 uL/min
Select the Channel for each compound
Load the 96 well plate according to the Plate Layout in the software
- Load the compounds to match each channel with immobilized protein
- Load solvent correction in the plate
- Load SPR running buffer in the unused wells
Dilute compounds two-fold in DMSO, starting at 100 uM top concentration in separate 96 well plate. Can fit 16 compounds in one plate with 6 points (The program will add a 7th point with 0 analyte as a control, this is just running buffer)
Note: Use plate foils to prevent evaporation during these longer runs, foils are not necessarily needed for any run under 1 hour (including immobilzation and HitID)
Select LMW - Multi Cycle Kinetics with wash
Set to run at 30 uL/min, 60s Association, 120s Dissociation. Ensure analyte is running over both flow cells
When designing plate layout, click settings icon in top right corner and check 'When plate is full, continue with next plate in same tray'
Note: Keep a record of vial numbers and corresponding experiment ID to add to Box folder
Organize plates so that each solvent correction step is in plate 1, and each plate has DMSO wash
Note: Be careful when adding compounds to their respective plates, often the system will have compounds 1-8 in plate 2 and 9-16 in plate 1. The machine will then start with plate 2 after solvent correction
Open hotel door and add plates to corresponding slots
Send method to queue, a run with 32 compounds will take about 3.5 hours.
Change chip back to maintenance chip
Insert buffer Line A back into water and perform change solution protocol
Perform desorb protocol in deep well plate
Note: Desorb solution 2 is kept refrigerated
When desorb is done, wash deep well plate and top off the water carefully, leave lines fully submerged in water
Close out of all methods and evaluations but can leave computer on
Open evaluation software and create a new evaluation
Select the run you wish to evaluate, then for the method select predefined methods, LMW, LMW Multi Cycle Affinity for equilibrium experiments or LMW Multi Cycle Kinetics/Affinity for kinetic experiments
Select sensorgrams and choose for the sensorgrams to be displayed by analyte (the default is channel)
Select affinity and quality check curves. May manually add Rmax where needed for representative curves
Note: Choose Rmax with lowest chi-squared, typically this is not the y-max (May eyeball this, if Rmax is manually added then the Kd is a rough estimation anyway and compound will need to be repeated if promising)
For export of data, return to home and choose export as spreadsheet
Save to flash drive, then close evaluation and save as new evaluation to the Chordoma Foundation folder
Curves should be smooth and generally reach equilibrium after 60s. Hits will have a higher RU and non-hits will be flat
Curves are not smooth and do not reach equilibrium. Large spikes present
Solutions: Bumpy curves are generally attributed to poor solubility, may need to repeat compound at a lower concentration
If curves are not reaching equilibrium, contact time may need to be extended to 90 or 120 seconds
Negative slopes and large spikes may be due to poor solvent matching, ensure careful pipetting of compounds and DMSO into running buffer
Curves are smooth and reach equilibrium and return to 0 after dissociation. Spacing of curves is indicative that saturation has been reached, which is confirmed by affinity curve. Affinity curve fitting is good and Kd is contained within the titration points. Rmax is within reason compared to the theoretical Rmax
Solution: Increase contact time This one is close to equilibrium at most points, so I would go to 90s instead of 60s
Poor separation of points, the distance between points should not continue increasing up to the highest concentration. This results in a more linear affinity curve, which is difficult to fit.
Solution: Can increase top concentration to obtain a more accurate Kd
Not reaching equilibrium, slow on time and slow off time
Solution: Increase the contact time in order to reach equilibrium, may also need to increase dissociation time. For these slower compounds, it is also possible to use a kinetic evaluation to calculate Kd from ka and kd
Very low Rmax, points out of solvent correction range
Solution: If the Rmax is significantly lower than the theoretical Rmax, it may mean that the compound is not binding. Repeat experiment with new immobilization
Points out of solvent correction range are usually due to problems with solubility, try decreasing top concentration and repeat experiment
