933 Methods
- Mix 10 μl of corneocyte extract and 70 μl of caspase-14 assay
buffer ( see Note 2 ). - Incubate for 15 min at room temperature.
- Add 10 μl of 0.2 mM Ac-WEHD-AFC (dissolved in DMSO)
and incubate for 30 min at 37 °C ( see Note 3 ). - Measure enzyme activity on plate reader with 355 nm excita-
tion and 460 nm emission. - Prepare 10–15 g of cornifi ed cells by scrapping them from
heels of healthy individuals. - Homogenize ca. 0.5 g in 20 ml of the extraction buffer using
glass homogenizer. - Centrifuge at 15,000 × g for 60 min. Remove supernatant and
concentrate to ca. 2 ml using protein concentrator. - Desalt the sample using Fast Desalting column equilibrated
with 20 mM Tris–HCl (pH 8.0), or dialyze against the same
buffer. - Apply the crude extract to a HiPrep 16/10 Q XL column,
wash the column with 20 mM Tris–HCl (pH 8.0) and elute
with a 0–1 M linear NaCl gradient. - Monitor hydrolytic activity against WEHD-AFC in each
fraction. - Concentrate active fraction(s) to 3.5 ml using protein
concentrator. - Exchange buffer with 20 mM Tris–HCl (pH 8.0) equilibrated
with 20 mM acetate buffer (pH 4.5). - Apply active fractions to a Mono Q column equilibrated with
the same buffer and elute with 0–1 M NaCl gradient. - After concentration and buffer-exchange, apply caspase-14-
containing fractions to Mono S cation-exchange column equil-
ibrated with 20 mM acetate buffer (pH 4.5) and elute with
0–1 M NaCl gradient. - After concentration and buffer-exchange, apply active fractions
to a chromatofocusing Mono P column equilibrated with
25 mM ethanolamine (pH 8.3). - Elute with 46 ml of Polybuffer (pH 5.0) forming a pH gradient
from pH 8.0 to pH 5.0 and fi nally with 2 M NaCl. - Apply caspase-14 containing fraction (less than 1 ml volume)
on Superdex 75 gel column equilibrated with PBS. - Determine protein concentration using, e.g., a Bio-Rad protein
assay kit.
3.1 Measurement
of Caspase-14 Activity
3.2 Purifi cation
of Caspase-14 from
Corneocyte Extract
Caspase-14 Methods