Caspases,Paracaspases, and Metacaspases Methods and Protocols

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15. Check protein quality on SDS-PAGE (5–15 % gradient gel)
    after staining with CBB ( see Fig. 1a ).


16. For further characterization, Western blot can be performed
    using the cleavage-site-directed antibody (h14D146) ( see
    Subheading 3.5 ), antibody to the large subunit (H99), and
    antibody to the small subunit of caspase-14 (C20) ( see Fig. 1b ).


17. Procaspase-14 cDNA is PCR cloned into the Bam HI/Kpn I
    sites of pQE 30 vector using forward primer cgggatccatgag-
    caatccgcggtctttgg and reverse primer ggggtaccctagatgac
    catcacaatctc.


18. Clone large and small subunits using the procaspase-14 con-
    struct as a template DNA. The construct consisting of small

3.3 Preparation
of Constitutively
Active Caspase-14
(revC14)

17 KDa

50K

a

b

35K

25K

15K

10K

30K

11 KDa

M C14 M C14 M C14

WB: H99 h14D146 C20

Fig. 1 Purifi cation and characterization of human caspase-14. ( a ) SDS-PAGE

analysis of purifi ed caspase-14. Human caspase-14 was purifi ed from corneo-

cyte extract with successive chromatographic procedures. The caspase-14

containing fraction collected after Superdex gel chromatography was subjected

to SDS-PAGE analysis. Gel was stained with CBB. The 17 kDa band corresponds

to the large subunit and the 11 kDa bands to the small subunit of caspase-14.

( b ) Western blot analysis of the purifi ed fraction. The purifi ed fraction was verifi ed

with three different antibodies to caspase-14: H99 (recognizes the large sub-

unit), h14D146 (recognizes cleavage site of the active caspase-14), and C20

(recognized the small subunit). M molecular weight marker, C14 purified

caspase-14 fraction

Mami Yamamoto-Tanaka and Toshihiko Hibino

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