From the compound you weigh to the published plate readings.
See how one compound's formula weights become a stock and a dilution plan, and how that plan sits beside a published screen's raw plate readings. The guided example goes on to fit a dose–response curve to those readings in Assay.
Molecule
Start with the compound you actually weigh.
The salt, erlotinib hydrochloride, has a formula weight of 429.90 g/mol; its parent, erlotinib, 393.44 g/mol. Weighing the salt by the parent's formula weight gives a stock 8.5% too weak.
Salt and parent formula weights - Salt formula weight
- 429.90 g/mol
- erlotinib hydrochloride
- Parent formula weight
- 393.44 g/mol
- erlotinib
Salt formula weight
Parent formula weight
- Shortfall if the parent's weight were used
- 8.5%
Formula weights from CIAAW 2021 atomic weights
Carried forward: both formula weights, 429.90 and 393.44 g/mol
Calc
Plan the stock and serial dilution.
Calculate a 5 mM stock and plan an 8-point, 4-fold dilution series from 10 µM to 610 pM, with pipetting-minimum flags.
Illustrative preparation plan · not measured results - Mass to weigh
- 4.299 mg
- of the salt, made up to 2 mL with DMSO for a 5 mM stock
- Dilution series
- 8 points
- 4-fold dilution
610 pM10 µM
In your plan, hold every well, vehicle included, at the top point's 0.2% DMSO.
Carried forward: 8 planned concentrations (10 µM → 610 pM)
Assay
Set your plan beside the published readings.
Compare your plan with the published series, which it reproduces, from PRISM Repurposing 19Q4. Inspect raw fluorescence across 3 plates without losing provenance.
Raw plate readings, before normalisation or fitting Raw median fluorescence intensity (MFI)
Published dose (logarithmic scale)
PREP016_CS5_X2 well C3 (10 µM): no reading in the source; shown as missing.
Broad Institute, PRISM Repurposing 19Q4, screen HTS002, HCC827 cells, pooled barcode MFI, CC BY 4.0, doi:10.6084/m9.figshare.9393293.v4
The Broad made these plates; this example did not.
Inspect the published readings
Read the source dataset ↗Plate Well Dose (µM) Raw MFI PREP016_CS5_X1 C3 10 931 PREP016_CS5_X1 C4 0.625 396 PREP016_CS5_X1 C13 2.5 396 PREP016_CS5_X1 C14 0.15625 475.5 PREP016_CS5_X1 D3 0.0390625 5907 PREP016_CS5_X1 D4 0.0024414 13879 PREP016_CS5_X1 D13 0.00976562 15212.5 PREP016_CS5_X1 D14 0.00061034 12118 PREP016_CS5_X2 C3 10 Missing PREP016_CS5_X2 C4 0.625 597 PREP016_CS5_X2 C13 2.5 616 PREP016_CS5_X2 C14 0.15625 456 PREP016_CS5_X2 D3 0.0390625 2012 PREP016_CS5_X2 D4 0.0024414 14351 PREP016_CS5_X2 D13 0.00976562 8626 PREP016_CS5_X2 D14 0.00061034 11038 PREP016_CS5_X3 C3 10 336 PREP016_CS5_X3 C4 0.625 335.5 PREP016_CS5_X3 C13 2.5 315 PREP016_CS5_X3 C14 0.15625 324.5 PREP016_CS5_X3 D3 0.0390625 3569.5 PREP016_CS5_X3 D4 0.0024414 14662 PREP016_CS5_X3 D13 0.00976562 14961 PREP016_CS5_X3 D14 0.00061034 15481.5
A walkthrough · Published plate readings
The plate readings were published by the Broad Institute (PRISM Repurposing 19Q4, CC BY 4.0); the guided example did not make the plates. The app estimates an IC50 in HCC827 cells; its profile interval’s upper bound is not resolved within the search. This is an example, not your measurement. What the example adds is labelled as an example and can be removed in one action.
