Contributors: Tank
First of six MUC/MUG plate-reader runs (19 Aug – 15 Sep 2026), all read on a BMG Omega, Greiner 96 F-bottom black-wall/clear-bottom plate, bottom optic, Ex 310-10 / Em 460. Figures below are re-derived from the raw data exports, not from notes.
Purpose
First MUC assay: CxnA pellet and culture supernatant, volume series 10 / 20 / 50 / 100 µl. Test ID 264. Gain 1000, 2 min × 627 cycles = 20.9 h, temperature not set.
Results
| Observation | Value | Meaning |
|---|---|---|
| 50 µl pellet reached a true plateau | net +127,789 FSU; terminal slope −4.25 FSU/min | 200 µM substrate fully converted — later reused as an in-matrix single-point calibrator |
| Volume-series efficiency | 10→50 µl linear (1 : 2 : 4.7); 100 µl collapsed to 4% | Attributed at the time to matrix quenching — later shown to be an artefact of changing volume, not enzyme concentration |
| Standard curve inverted | F1 (labelled 400 µM) read 460; F11 (labelled 0 µM) read 2,877 | Series was loaded in the opposite direction to the record |
| Standards drifted upward | F7: 1,466 → 11,792 over 21 h | 4-MU injected from DMSO into PBS hadn’t fully dissolved; still dissolving at 21 h |
| Dead well | A4: 534 → 631, flat | No sample added |
Problems and actions
- Standard curve unusable → prepare the dilution series in DMSO, not PBS; drop the top concentration.
- Loading direction not recorded → mark an arrow on the plate edge going forward.
- 22 h read → substrate exhausted; shorten read time.
- Protein by A280 gave impossible values (0.247 → reported as 247 mg/mL, from the 18 August prep) → confirmed as a unit error; A280 on crude lysate is unreliable anyway (nucleic acid contribution).