Contributors: Tank
Set up the first MUC (4-methylumbelliferyl β-D-cellobioside, exoglucanase substrate) baseline assay for CxnA.
Culture and lysis
Overnight induced cultures (0.38 mM IPTG, 40 µg/mL chloramphenicol): CxnA in Top10, and CxnA-His in Top10 (frameshifted — non-expressing control). Cells were pelleted, supernatant separated, and the pellet resuspended in 5 mL BugBuster + ½ tablet protease-inhibitor (EDTA-free, Roche).
Protein concentration (A280)
| Sample | A280 | Reported |
|---|---|---|
| CxnA supernatant | 0.247 | recorded as “247 mg/mL” |
| CxnA-His supernatant | 0.179 | recorded as “179 mg/mL” |
| CxnA pellet (1:20 dilution) | 0.285 | 5.7 mg/mL |
| CxnA-His pellet (1:20 dilution) | 0.235 | 4.7 mg/mL |
The supernatant values are almost certainly a units error (absorbance read directly as mg/mL without applying an extinction-coefficient conversion) rather than real concentrations — a crude-lysate A280 reading is unreliable for absolute protein quantitation anyway, given nucleic acid contribution to the same wavelength. This gets formally flagged and corrected in the 19 August assay write-up.
96-well plate loading
MUC stock at 20 mM; excitation 365 nm / emission 455 nm. Loaded a volume series of CxnA/CxnA-His pellet and lysate (10/20/50/100 µl) against PBS + MUC, plus a 4-MU standard series (0–400 µM) in row F for calibration.
Note: the MUC stock ran out partway through loading (from well D10 onward) — data from D10 through E11 is not usable.
This plate’s read became the first formal MUC assay run, written up under 19 August.