APOPTOSIS

APOPTOSIS INDUCERS

The experimental manipulation of apoptosis has been greatly facilitated by the identification of a large variety of pharmacological tools which can induce apoptosis via diverse mechanisms. The agents listed below are capable of inducing apoptosis by a variety of mechanisms including Ca²⁺ influx, oxidative stress, Zn²⁺ chelation, cytoskeletal perturbation, inhibition of protein synthesis, perturbation of DNA integrity and alteration of gene expression as well as by undefined mechanisms.


TABLE 9. APOPTOSIS INDUCING REAGENTS

AP-301
Betulinic acid
98% MW=456.7 [472-15-1] Storage: -20°C
Betulinic acid is a selective inhibitor of human melanoma which functions by induction of apoptosis²⁷. Anti-HIV activity^28,29.
20 mg
100 mg

AP-300
TPEN
N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine
98%, MW=424.6 [16858-02-9] Storage: RT
Useful tool for chelation of intracellular Zn²⁺. Has low affinity for Ca²⁺ and Mg²⁺. Cell permeable. Induces apoptosis in T cells^30,31.
100 mg

PARP ENZYME

SE-165
PARP enzyme (from bovine spleen)
Poly(ADP-ribose) polymerase (EC 2.4.2.30)
30-50% by SDS-PAGE, MW=116 kDa, Storage: -70°C
Bovine PARP purified by DNA-cellulose affinity chromatography. Supplied in sucrose buffer. Specific activity is approximately 200 nmol ADP-ribose/min/mg total protein at 25°C. Applications: i) Standards for Western blotting (50-100 ng/lane) using anti-PARP (BIOMOL catalog #SA-249, -250, -251, -252)^32,33 . ii) Enzyme automodified with NAD can be used as WB standard for anti-poly(ADP-ribose) (BIOMOL catalog #SA-216, -217) and activity gels^33,34. iii) To synthesize poly(ADP-ribose) polymer for use in ELISA assays for autoimmune disease³⁵, polymer binding proteins and for glycohydrolase assays³³. iv) Substrate for CPP32 (apopain) or other proteases such as granzyme B³⁶.
100 µg

PARP INHIBITORS

G-400
3-Aminobenzamide
97%, MW=136.2 [3544-24-9] Storage: RT
PARP inhibitor. Inhibits nitric oxide-induced apoptosis but not differentiation in HL-60 cells³⁷. Protects cells from oxygen radical and nitric oxide toxicity³⁸. Inhibits stress-induced apoptosis³⁹.
1g
5g

AP-101
4-Amino-1,8-naphthalimide
96%, MW=212.2 [1742-95-6] Storage: RT
PARP inhibitor. Reduces ischemia-reperfusion injury in the heart and skeletal muscle⁴⁰.
20 mg
100 mg

AP-100
6(5H)-Phenanthridinone
98%, MW=195.2 [1015-89-0] Storage: RT
PARP inhibitor. Displays immunosuppressive activity. Inhibits concanavalin A-induced lymphocyte proliferation at micromolar concentrations⁴¹.
200 mg

PARP ANTIBODIES

SA-250
Anti-poly(ADP-ribose) polymerase (PARP)
Mouse monoclonal antibody. Clone C-2-10. Immunogen: Purified calf thymus poly(ADP-ribose) polymerase (PARP). Supplied as ascites IgG1. The epitope is in the C-terminal part of the DNA binding domain of PARP^42,43. Recognizes 116 kDa PARP and the 85 kDa apoptosis-related cleavage fragment. Crossreacts with human, monkey, hamster, rat and mouse PARP. Applications: WB 1/2000, IC 1/1000 and ELISA.
50 µl

SA-249
Anti-poly(ADP-ribose) polymerase (PARP)
Mouse monoclonal antibody. Clone C-2-10. See SA-250, above.
Supplied as purified IgG1, 0.2 mg/ml in PBS. Applications: WB 1 µg/ml, IC and ELISA.
100 µg

SA-251
Anti-poly(ADP-ribose) polymerase (PARP)
Mouse monoclonal antibody. Clone F1-23. Immunogen: Purified calf thymus poly(ADP-ribose) polymerase (PARP)^42,43. Supplied as ascites IgG1. The epitope is in the zinc finger II DNA binding domain of PARP. Detects 116 kDa PARP and the 24 kDa apoptosis-related cleavage fragment. Recognizes human, monkey and bovine PARP. It does not crossreact with rat or mouse. Applications: WB 1/500 (using ECL).
25 µl

SA-252
Anti-poly(ADP-ribose) polymerase (PARP), C-terminal
Rabbit polyclonal antibody. Immunogen: Synthetic peptide based on bovine PARP automodification domain. Supplied as serum. Recognizes 116 kDa PARP and the 85 kDa apoptosis-related cleavage fragment^42,43,44. Crossreacts with human, monkey, hamster, rat, mouse and chicken PARP. Applications: WB 1/5000 (using ECL) and ELISA
100 µl

SW-101
HL60 Cell Extract, Noninduced
Immunoblotting standard
Whole cell extract of human HL60 leukemia cells⁴⁵. Supplied in SDS-PAGE sample buffer, ready to use. Applications: WB control for SA-250 and SA-252, above, to detect intact 116 kDa PARP. Recommended usage is 20 µl per lane (~75,000 cells). Storage: -20°C
200 µl

SW-102
HL60 Cell Extract, Induced
Immunoblotting standard
Whole cell extract of human HL60 leukemia cells, induced to undergo apoptosis by the chemotherapeutic agent etoposide⁴⁵. Supplied in SDS-PAGE sample buffer, ready to use. Applications: WB control for SA-250 and SA-252, above, to detect PARP cleavage. Recommended usage is 20 µl per lane (~75,000 cells). Storage: -20°C
200 µl

POLY(ADP-RIBOSE), POLYMER

SA-216
Anti-poly(ADP-ribose)
Mouse monoclonal antibody. Immunogen: Purified poly(ADP-ribose) polymer, chain length of 10-50 units⁴⁶. Supplied as mouse ascites IgG3 kappa. Recognizes poly(ADP-ribose) modified proteins independent of species source^47,48. There is no crossreactivity with RNA, DNA, monomers of ADP-ribose or NAD. Application: WB 1/500, IP and IH.
100 µl

SA-217
Anti-poly(ADP-ribose)
Guinea pig polyclonal antibody. Immunogen: Purified poly(ADP-ribose) polymer. Supplied as guinea pig serum. Recognizes poly(ADP-ribose) modified proteins independent of species source. Application: WB 1/500 and IP.
100 µl

SW-108
Automodified PARP
Immunoblotting standard
Partially purified PARP enzyme, SE-165, poly (ADP-ribose) automodified by the addition of NAD and necessary cofactors. Supplied in SDS-PAGE sample buffer, ready-to-use. A streak of MW >116 kDa is detected by Western blotting, which represent PARP modified to various extents by poly(ADP-ribose). Applications: WB control for SA-216 and SA-217, above. Recommended usage is 5-20 µl per lane. Storage: -20°C.
100 µl

SW-100
Poly(ADP-ribose) protein
Immunoblotting standard
Crude protein extract of E. coli expressing human poly(ADP-ribose) polymerase (PARP). Supplied as 0.5 mg/ml protein in SDS-PAGE sample buffer, ready-to-use. Several protein bands representing poly(ADP-ribose) modified PARP in addition to modified E. coli proteins are detected by Western blotting. Applications: WB control for SA-216 and SA-217, above. Recommended usage is 5-10 µg per lane. Storage: -20°C
25 µg

CPP32 (CASPASE-3/CPP32b/APOPAIN/YAMA)

CPP32 is a member of a family of cysteine proteases that include IL-1 b-converting enzyme (ICE), Ich-1 (Nedd2) and others. The proteases of this group have been implicated as regulators of programmed cell death or apoptosis^49,50. CPP32, most related to Ced-3 (from C. elegans), is also known as apopain, prICE and Yama^51,52,53. CPP32 is distinguished by its ability to cleave poly(ADP-ribose) polymerase (PARP) to generate a 85 kDa fragment during apoptosis. The cleavage site in PARP is C-terminal to Asp-216⁵³, a site conserved in human, bovine and chick PARP. The upstream sequence, DEVD, is the basis for the extremely specific peptide substrates, inhibitor and affinity reagent described below.

P-411
DEVD-AMC, CPP32 fluorogenic substrate (AMC)
N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin)
>97%, MW=694.0 Storage: -20°C
Sequence is based on PARP cleavage site Asp-216 for CPP32^51,52. Fluorescence assay shows linear Michaelis-Menton kinetics with a K_m=9.7 mM⁵¹. Excitation: 380 nm, Emission.: 460 nm.
5 mg
5 x 5 mg

P-409
DEVD-AFC, CPP32 fluorogenic substrate (AFC)
N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl coumarin)
>97%, MW=729.0 Storage: -20°C
Similar to P-411 but the AFC fluorophore has a greater Stokes' shift upon cleavage. Reaction can be monitored quantitatively or visually by a blue to green shift in fluorescence upon cleavage. Excitation: 400nm, Emission: 505nm.
5 mg
5 x 5 mg

P-412
DEVD-pNA, CPP32 colorimetric substrate (PNA)
N-acetyl-Asp-Glu-Val-Asp-pNA (p-nitroanilide)
>97%, MW=638.0 Storage: -20°C
Similar to P-411 but cleavage is monitored colorimetrically by absorbance at 400 nm.
5 mg
5 x 5 mg

P-410
DEVD-CHO, CPP32 inhibitor (CHO)
N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)
>95%, MW=502.5 Storage: -20°C
Highly specific, potent and reversible inhibitor of CPP32⁵¹. K_i <1 nM for purified CPP32. IC₅₀=0.2 nM for PARP cleavage in cultured human osteosarcoma cell extracts. In contrast, the ICE-specific inhibitor YVAD-CHO (BIOMOL catalog #P-403) has a K_i =12 µM for CPP32 and IC₅₀>10 µM for PARP cleavage in osteosarcoma cell extracts⁵¹.
1 mg
5 x 1 mg

P-413
Biotin-DEVD-CHO, CPP32 affinity reagent
N-biotin-Asp-Glu-Val-Asp-CHO (aldehyde)
>90%, MW=686.8 Storage: -20°C
Highly specific, affinity reagent for CPP32⁵¹. Useful for the purification or extract depletion of CPP32 in conjunction with streptavidin conjugates.
1 mg
5 x 1 mg

P-414
Cell-Permeable DEVD-CHO, CPP32 inhibitor
>97%, MW=2000.4, Storage: -20°C
This unique 20 amino acid polypeptide is composed of two domains. The C-terminal DEVD-CHO region is a highly specific, potent and reversible inhibitor of CPP32. K_i<1 nM for purified CPP32. IC₅₀=0.2 nM for PARP cleavage in cultured human osteosarcoma cell extracts. The DEVD sequence is based on PARP cleavage site Asp-216 for CPP32^51,52. The N-terminal sequence (residues 1-16) has been shown to facilitate cell-permeability of peptides^54,55. It is based on the hydrophobic region (h-region) of the signal peptide of Kaposi fibroblast growth factor (K-FGF). This peptide has the potential to be a useful cell-permeable CPP32 inhibitor.
Inquire

IL-1 b-CONVERTING ENZYME (ICE/CASPASE-1)

P-400
IL-1 b-converting enzyme (ICE) substrate
Asn-Glu-Ala-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-Ser-Leu-Asn
>97%, MW=1584.8 Storage: -20°C
Sequence is precursor interleukin-1 b (110-123) inclusive of cleavage site (C-terminal to Asp-116)^56,57. Peptide is a substrate and competitive inhibitor of ICE⁵⁸
1 mg
5 x 1 mg

P-401
YVAD-AMC, ICE fluorogenic substrate (peptide 17)
N-acetyl-Tyr-Val-Ala-Asp-AMC (7-amino-4-methylcoumarin)
>97%, MW=665.7 Storage: -20°C
Sequence is based on precursor interleukin-1 b Asp-116 cleavage site for ICE^57,58. Fluorescence assay shows linear Michaelis-Menton kinetics with a K_m =14 µM^56,59. Excitation: 380 nm, Emission: 460 nm⁶⁰.
5 mg
5 x 5 mg

P-406
Z-YVAD-AFC, ICE fluorogenic substrate (AFC)
Z-Tyr-Val-Ala-Asp-AFC (7-amino-4-trifluoromethyl coumarin)
>95%, MW=811.8 Storage: -20°C
Similar to P-401 but the AFC fluorophore has a greater Stokes' shift upon cleavage, and the N-terminal is blocked by a benzyloxycarbonyl (Z) moiety which increases cell permeability. Reactions can be monitored quantitatively or visually by a blue to green shift in fluorescence upon cleavage. Excitation: 400 nm, Emission: 505 nm.
5 mg
5 x 5 mg

P-408
YVAD-pNA, ICE colorimetric substrate (PNA)
N-acetyl-Tyr-Val-Ala-Asp-pNA (p-nitroanilide)
>97%, MW=628.6 Storage: -20°C
Similar to P-401 but cleavage is monitored colorimetrically by absorbance at 400 nm^60,61.
5 mg
5 x 5 mg

P-403
YVAD-CHO, ICE inhibitor (CHO)
N-acetyl-Tyr-Val-Ala-Asp-CHO (aldehyde)
>97%, MW=492.0 Storage: -20°C
Potent, reversible inhibitor of ICE. K_i=1 nM⁶².
1 mg
5 x 1 mg

P-404
Cell-Permeable YVAD-CHO, ICE inhibitor
>97%, MW=1990.4, Storage: -20°C
The YVAD-CHO portion of this peptide is a highly specific, potent and reversible inhibitor of ICE. K_i=1 nM for purified ICE. Sequence is based on precursor interleukin-1b Asp-116 cleavage site for ICE^56,62. The N-terminal portion of this peptide contains a cell permeable sequence identical to that of P-414^54,55. This peptide has the potential to be a useful cell-permeable ICE inhibitor.
INQUIRE

OTHER ANTIBODIES

SA-239
Anti-Bcl-2, human
Mouse monoclonal antibody. Clone 4D7. Immunogen: Human bcl-2a (61-76) peptide conjugated to carrier. Supplied as purified IgG1. Recognizes 25-26 kDa human bcl-2 protein. Does not crossreact with bcl-x or bax protein⁶³. Applications: WB 1 µg/ml, IP, IF.
100 µg

SA-190
Anti-Bcl-2, human
Hamster monoclonal antibody. Clone 6C8. Immunogen: Human bcl-2 recombinant protein expressed in E. coli⁶⁴. Supplied as purified IgG in TBS/sodium azide. Recognizes p25 kD human bcl-2, with no cross-reaction to mouse^65,66. Applications: WB 0.5-2 µg/ml, IP, IH, IF.
100 µg

SA-191
Anti-Bcl-2, mouse
Hamster monoclonal antibody. Clone 3F11. Immunogen: Mouse bcl-2 recombinant protein expressed in E. coli⁶⁷. Supplied as purified IgG in TBS/sodium azide. Recognizes p25 kD mouse bcl-2, with no cross-reaction to human⁶⁸. Applications: WB 5 µg/ml, IP, IH, IF.
100 µg

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