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12675
Initiator Caspases Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Initiator Caspases Antibody Sampler Kit #12675

Citations (1)
Simple Western™ analysis of lysates (1 mg/mL) from Jurkat cells treated with Cytochrome C using Caspase-3 (D3R6Y) Rabbit mAb #14220. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of various cell lines, untreated (-) or treated with Staurosporine #9953 (1 μM; 3 hr) or with Etoposide #2200 (25 μM, overnight), using Caspase-3 (D3R6Y) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). MCF7 cells are negative for caspase-3 expression.
Western blot analysis of extracts from Jurkat cells, untreated or etoposide-treated (5 hr, 25μM), using Caspase-2 (C2) Mouse mAb.
Western blot analysis of extracts from HeLa and Jurkat cells, untreated (-) or treated with Staurosporine #9953 (1 μM, 3 hr; +) or Etoposide #2200 (25 μM, overnight; +), using Cleaved Caspase-9 (Asp330) (E5Z7N) Rabbit mAb (upper), Caspase-9 (C9) Mouse mAb #9508 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from Jurkat cells (human), L929 cells (mouse), and C6 cells (rat), untreated or treated with staurosporine or cytochrome c as indicated, using Caspase 9 (C9) Mouse mAb.
Western blot analysis of extracts from control HeLa cells (lane 1) or Caspase-8 knockout HeLa cells (lane 2) using Caspase-8 (1C12) Mouse mAb #9746 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the Caspase-8-knockout HeLa cells confirms specificity of the antibody for Caspase-8.
Western blot analysis of extracts from HCT116 cells (lane 1) or CASP3 knock-out cells (lane 2) using Caspase-3 (D3R6Y) Rabbit mAb #14220 (upper), and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). The absence of signal in the CASP3 knock-out HCT116 cells confirms specificity of the antibody for CASP3.
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine #9953 (1 μM, 3 hr; right), using Cleaved Caspase-9 (Asp330) (E5Z7N) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from HCT 116 and HCT 116 Caspase-8 knockout cells, untreated (-) or treated with staurosporine #9953 (1 μM, 4 hr; +), using #9746 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the HCT 116 knockout cells confirms the specificity of the antibody for Caspase-8.
Western blot analysis of extracts from SKW6.4 cells, untreated or anti-Fas-treated (1 µg/ml), and Jurkat cells, untreated or etoposide-treated (25 µM), using Caspase-8 (1C12) Mouse mAb.
To Purchase # 12675
Cat. # Size Qty. Price
12675T
1 Kit  (5 x 20 microliters)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Cleaved Caspase-9 (Asp330) (E5Z7N) Rabbit mAb 52873 20 µl
  • WB
  • IF
H 37 Rabbit IgG
Caspase-3 (D3R6Y) Rabbit mAb 14220 20 µl
  • WB
  • IP
H M R Mk 35, 19, 17 Rabbit IgG
Caspase-2 (C2) Mouse mAb 2224 20 µl
  • WB
H 12, 14, 48 Mouse IgG1
Caspase-9 (C9) Mouse mAb 9508 20 µl
  • WB
H M R Hm Mk 47/37/35 (H). 49/39/37 (M). 51/40/38 (R). Mouse IgG1
Caspase-8 (1C12) Mouse mAb 9746 20 µl
  • WB
  • IP
H 18, 43, 57 Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

The Initiator Caspases Antibody Sampler Kit provides an economical means of evaluating initiator (apical) caspase proteins. The kit contains enough primary antibody to perform two western blots with each primary antibody.

Specificity / Sensitivity

Each antibody in the Initiator Caspases Antibody Sampler Kit recognizes its respective target at endogenous levels. Caspase-3 (D3R6Y) Rabbit mAb detects full-length caspase-3 (35 kDa) as well as the large subunit (p20) of caspase-3 resulting from cleavage during apoptosis. Caspase-8 (1C12) Mouse mAb recognizes full length (57 kDa), the cleaved intermediate p43/p41, and the p18 fragment of caspase-8. Caspase-9 (C9) Antibody recognizes full-length caspase-9, as well as the large fragments resulting from cleavage at Asp315 and Asp330. Cleaved Caspase-9 (Asp330) (E5Z7N) Rabbit mAb recognizes caspase-9 protein only when cleaved at Asp330. Caspase-2 (C2) Mouse mAb recognizes procaspase-2, as well as its 14 and 12 kDa cleaved fragments.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a recombinant protein specific to either the p20 subunit of human caspase-3 protein, or human caspase-9 protein. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp330 of human caspase-9, the carboxy-terminal sequence of the p18 fragment of human caspase-8, or the carboxy-terminal portion of human caspase-2.

Background

Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 2, 8, 9, 10 and 12) are closely coupled to proapoptotic signals, which include FasL, TNF-α, and DNA damage. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins such as PARP, α-fodrin, DFF and lamin A; inducing apoptosis (1,2).
Formation of a death-inducing signaling complex (DISC) around the receptors for death factors, including FasL and TNF-α, is essential for receptor-mediated apoptosis (3). Upon ligand activation, Fas and TNF-R1 associate with death domain (DD) containing adaptor proteins FADD (Fas associated death domain) (4,5) and TRADD (TNF-R1 associated death domain) (6). In addition to a carboxy-terminal DD, FADD contains an amino-terminal death effector domain (DED) that binds to DEDs and activates initiator caspase 8 (FLICE, Mch5, MACH) and caspase 10 (FLICE2, Mch4) (7-12). TRADD does not contain a DED and therefore must associate with FADD in response to TNF-R1 driven apoptosis (13).
Caspase-9 (ICE-LAP6, Mch6) is activated through the mitochondrial-mediated pathway. Cytochrome c released from mitochondria associates with procaspase-9 (47 kDa)/Apaf-1. Apaf-1 mediated activation of caspase-9 involves proteolytic processing resulting in cleavage at Asp315 and producing a p35 subunit. Another cleavage occurs at Asp330 producing a p37 subunit that can amplify the apoptotic response (14-17).
Caspase-2 (Nedd2/ICH-1) is the nuclear apoptotic respondent to cellular genotoxic stress or mitotic catastrophe. The procaspase is cleaved at Asp316, producing a 14 kDa fragment and a 32 kDa prodomain/large subunit. Subsequent processing at Asp152 and Asp330 produces an 18 kDa large subunit and a 12 kDa small fragment (18). Activation occurs upon recruitment to a complex containing a p53-induced death domain protein, PIDD (19). This suggests that caspase-2 can be a nuclear initiator caspase with a requirement for caspase-9 and caspase-3 activation in downstream apoptotic events (20,22). In apoptotic pathways resulting from UV-induced DNA damage, processing of caspase-2 occurs downstream of mitochondrial dysfunction and of caspase-9 and caspase-3 activation, extending a possible role for caspase-2 as a parallel effector caspase (22).
Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis and caspase-3 cleavage is a key indicator of initiator caspase activation. Caspase-3 is either partially or totally responsible for the proteolytic cleavage of many key proteins including the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (23). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments (24).

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  2. Cohen, G.M. (1997) Biochem J 326 ( Pt 1), 1-16.
  3. Nagata, S. (1997) Cell 88, 355-65.
  4. Chinnaiyan, A.M. et al. (1995) Cell 81, 505-12.
  5. Boldin, M.P. et al. (1995) J Biol Chem 270, 7795-8.
  6. Hsu, H. et al. (1995) Cell 81, 495-504.
  7. Muzio, M. et al. (1996) Cell 85, 817-27.
  8. Boldin, M.P. et al. (1996) Cell 85, 803-15.
  9. Vincenz, C. and Dixit, V.M. (1997) J Biol Chem 272, 6578-83.
  10. Fernandes-Alnemri, T. et al. (1996) Proc Natl Acad Sci U S A 93, 7464-9.
  11. Kischkel, F.C. et al. (2001) J Biol Chem 276, 46639-46.
  12. Wang, J. et al. (2001) Proc Natl Acad Sci U S A 98, 13884-8.
  13. Hsu, H. et al. (1996) Cell 84, 299-308.
  14. Liu, X. et al. (1996) Cell 86, 147-57.
  15. Li, P. et al. (1997) Cell 91, 479-89.
  16. Zou, H. et al. (1999) J Biol Chem 274, 11549-56.
  17. Srinivasula, S.M. et al. (1998) Mol Cell 1, 949-57.
  18. Li, H. et al. (1997) J Biol Chem 272, 21010-7.
  19. Tinel, A. and Tschopp, J. (2004) Science 304, 843-6.
  20. Dirsch, V.M. et al. (2004) Oncogene 23, 1586-93.
  21. Castedo, M. et al. (2004) Oncogene 23, 4362-70.
  22. Paroni, G. et al. (2001) J Biol Chem 276, 21907-15.
  23. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
  24. Nicholson, D.W. et al. (1995) Nature 376, 37-43.

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