CD47-SIRPalpha protein protein interaction - LANCE assay qHTS validation
- Deposit:2019-06-17
- Modify:2019-12-12
PROTOCOL TABLE (as described by Miller, T. et al. 2019)
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1a. Reagent. 3 uL CD47-His (12.5 nM) and Anti-His Eu Donor reagent (3 nM) added to respective wells in Assay buffer; white/solid bottom high base, med bind 1536-well plates (Greiner 789075)
1b. Reagent. 2 uL Assay buffer negative control for background fluorescence
2. Reagent. 23 nL library compounds (7-pt, 1:4 intraplate titration series) or DMSO control transferred by Pin Tool
3. Centrifuge. Centrifuge plate for 30 s at 1000 RPM
4. Incubation.#Incubate reagents at room temperature for 30 min, protected from light
5. Reagent. 2 uL SIRPalpha-biotin (100 nM) in Assay buffer
6. Incubation.#Incubate reagents at room temperature for 30 min, protected from light
7. Reagent. 1 uL SAV-APC (26 nM) acceptor reagent added to respective wells
8. Centrifuge. Centrifuge plate for 30 s at 1000 RPM
9. Incubation. Incubate reagents at room temperature for 30 min, protected from light
10. Detector. Measure Top TR-FRET on EnVision (Eu/APC filter set); calculate ratio Ch1/Ch2
Notes
1. Assay buffer composition.#Phosphate buffered saline (PBS; 10 mM Phosphate, 137 mM NaCl, and 2.7 mM KCl; pH 7.4), 0.05% IGEPAL CA-630
2. 23nL of LOPAC128 library compounds (7 plates) or DMSO vehicle control transferred to 9 Greiner white/solid bottom high base, TC plates in 7 interplate 1:5 titrations (see Yasgar, A. et al. 2008) with Pintool. Two of the 9 plates are DMSO-only in compound field at beginning and end of compound library-treated plates.
10. Detector settings#Mirror = LANCE/DELFIA Dual Enh; Ex. Filter = UV2[TRF] 320; Em. Filter = APC 665; 2nd Em. Filter = Europium 615; Measurement h = 6.5; Ex. light (%) = 100; Delay = 60 us; Window t = 100 us; No. Seq. windows = 1; Time between flashes = 2000us; No. of flashes = 100; No. of flashes for 2nd detector = 100; Calculation: "Ratio Ch1/Ch2"
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10.