Quantification of active caspase 3 in apoptotic cells.
We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the biotinylated inhibitor covalently bound to caspase 3. Using the assay we detected 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurkat cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biotinylated inhibitor. Specificity was also demonstrated by lack of signal with apoptotic MCF-7 cells which do not express caspase 3. The ability to discriminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was captured it was not covalently modified with the biotinylated inhibitor. The basic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has potential for quantifying active members of many classes of enzymes.
Saunders
P A
PA
R & D Systems, Inc., Minneapolis, Minnesota 55413, USA.
Cooper
J A
JA
Roodell
M M
MM
Schroeder
D A
DA
Borchert
C J
CJ
Isaacson
A L
AL
Schendel
M J
MJ
Godfrey
K G
KG
Cahill
D R
DR
Walz
A M
AM
Loegering
R T
RT
Gaylord
H
H
Woyno
I J
IJ
Kaluyzhny
A E
AE
Krzyzek
R A
RA
Mortari
F
F
Tsang
M
M
Roff
C F
CF
D017209:Apoptosis; D019921:Biotinylation; D053455:Caspase 10; D053143:Caspase 2; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D011994:Recombinant Proteins / Q000378:metabolism; D016622:Silver Staining; D019311:Staurosporine / Q000494:pharmacology; D019809:Streptavidin / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D020298:U937 Cells; D019014:fas Receptor / Q000276:immunology
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019014:fas Receptor; D019809:Streptavidin; D006735:Horseradish Peroxidase; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053455:Caspase 10; D053143:Caspase 2; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C506438:CASP10 protein, human; D019311:Staurosporine