96
- After extensive washing with PBS, monospecifi c antibody is
eluted with fi ve 0.5 ml aliquots of 0.1 M glycine-HCl buffer
(pH 2.3) and immediately neutralized with 0.5 ml of 1 M
Tris–HCl (pH 8.0). - Measure absorbance at 280 nm in each fraction and calculate
IgG concentration in mg/ml using formulae A280 × 0.714. - Exchange buffer to PBS using PD-10 column equilibrated
with PBS or dialyze against PBS. Store in small aliquots at
−80 °C.
Cleavage-site-directed antibody (h14Y178) is prepared by immu-
nizing rabbits with synthetic hexapeptide CGYIAY, in which
GYIAY corresponds to the C-terminal end of p20 cleaved by
KLK7. h14Y178 can be purifi ed using above described procedure
for h14D146.It is important to distinguish zymogen from the active form of
caspase-14, since proteolytic activation is a critical event for physi-
ological action of caspase-14. Here we describe two kinds of ELISA
quantifi cation assays using H99 antibody directed to the large
subunit of human caspase-14 (to measure total caspase-14) and
cleavage- site-directed antibody, h14D146 (to measure active
caspase- 14). We used in-house monoclonal anti-caspase-14 anti-
body, clone 3, as an immobilized antibody that showed the highest
sensitivity in the ELISA system. However, commercially available
mouse monoclonal antibody to caspase-14 can also be used.- Immobilize 1 μg/ml of anti-caspase-14 (clone 3 or commer-
cially available monoclonal antibody) in PBS by overnight
incubation in a 96-well plate. - Tap off the solution from the plate.
- Block by adding 200 μl/well of ImmunoBlock diluted with
PBS and incubate for 1 h at room temperature. - Tap off the solution from the plate and wash each well with
200 μl of PBS containing 0.1 % Tween 20 (PBST). - Add 100 μl samples (cell extracts) and incubate at 37 °C for
1 h. Use recombinant procaspase-14 as a standard to quantify
total caspase-14. Use revC14 as a standard to quantify active
caspase-14. - Wash the wells three times with 200 μl of PBST.
- For measurement of total caspase-14, add 2 μg/ml of H99
antibody in 1:20 diluted ImmunoBlock and incubate for 1 h at
37 °C. The active form of caspase-14 is measured in a similar
way using h14D146 as a primary antibody instead of H99.
3.5.2 Cleavage-Site-
Directed Antibody
(h14Y178)
3.6 ELISA Assays
for Caspase-14
Mami Yamamoto-Tanaka and Toshihiko Hibinohttp://www.ebook3000.com