Caspases,Paracaspases, and Metacaspases Methods and Protocols

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89 Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols, Methods in Molecul ...

90 fi bers embedded in fi laggrin to form macrofi brils. They also gain a reinforced, cytoplasmic membrane called CE, wherein om ...

91 completely different from that of caspase-14, showing broad specifi city for various caspase substrates except WEHD-AFC, the ...

92 Extraction buffer: 0.1 M Tris–HCl (pH 8.0), 0.14 M NaCl, and 1 mM phenylmethane sulfonyl fl uoride. 20 mM Tris–HCl (pH 8.0). ...

93 3 Methods Mix 10 μl of corneocyte extract and 70 μl of caspase-14 assay buffer ( see Note 2 ). Incubate for 15 min at room t ...

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

95 subunit fused to the N-terminal of the large subunit, ending at D^146 , makes constitutively active caspase-14. Insert revC1 ...

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 (p ...

97 Wash the wells three times with 200 μl of PBST. Add ECL™ Anti-rabbit IgG Horseradish Peroxidase linked F(ab)′ 2 fragment (GE ...

98 Ab: h14D146 Procaspase-14 concentration (μg/ml) 0 0.05 0.1 0.15 0.2 0.25 0.05 0.1 0.15 Absorbance at 405 nm Ab: h14D146 R^2 = ...

99 4 Notes For the measurement of revC14-Y178, sodium citrate can be omitted. Caspase-14 requires high concentration of kosmotr ...

100 References Eckhart L, Ban J, Fischer H, Tschachler E (2000) Caspase-14: analysis of gene structure and mRNA expression duri ...

101 Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols, Methods in Molecu ...

102 The central cell-killing pathway in C. elegans (EGL-1 –| CED-9 –| CED-4 → CED-3) is well characterized [ 4 ]. CED-3 acts at ...

103 Syringes and 0.45 μm fi lters. Ni-NTA beads (e.g., Qiagen catalog number 1018244). Sonication buffer: 100 mM Tris–HCl pH 8. ...

104 3 Methods Transform BL21 (DE3) with the CED-3-expressing con- struct, inoculate a single transformed colony in 10 ml of LB ...

105 As a negative control, the same concentration of DMSO as in the reaction solution can be used, as the substrate has been di ...

106 substrate, site-directed mutagenesis of the predicted CED-3 cleavage sequence is usually performed. Asp to Glu substitution ...

107 Stain the membrane with the Ponceau S solution at room tem- perature for 5 min and wash with destaining buffer for 10 min. ...