rev:   March 10, 2004

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  ANTIBODIES  

                                                      (anti-Human and others as indicated)
RDI Divison of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.


anti-CPP32 (CASPASE-3) Antibodies

Rabbit (monoclonal) anti-caspase-3 Active form (human/mouse)

cat#RDI-CASP3ACTabRm2      $410.00/50ug in PBS with 5% trehalose

Package: 1mg in 1 ml PBS 5% trehalose

Specificity: The  antibody specifically recognizes the active form of caspase-3 in human and mouse cells. It detects the p17 subunitt of Caspase 3 but does not detect the precursor form. 

Use:

-histochemistry: 0.1-0.5 ug.ml

Optimal dilution should be determined by the researcher.

Background: The caspase family of cysteine proteases plays a key role in apoptosis and inflammation (reviewed in 1). Caspase-3 (CPP32, Yama, apopain) is a key protease that is activated during the early stages of apoptosis and, like other members of the caspase family, is synthesized as an in- active proenzyme that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another protease. The processed forms of caspases consist of large (17-22 kD) and small (10-12kD) subunits which associate to form an active enzyme. Active caspase-3, a marker for cells undergoing apoptosis, consists of a heterodimer of 17 and 12 kD subunits which is derived from the 32 kD proenzyme.2 Active caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the cyto- plasm, e.g.,D4-GDI and Bcl-2(3). and in the nucleus, e.g. PARP.

 

For Research Use Only


rabbit anti-caspase-3-active form (human)

cat#RDI-CASP3ACTabr   $406.00/100ug in 0.2ml

cat#RDI-CASP3ACTab-BT Biotin labeled   $406.00/100ug

cat#RDI-CASP3ACTab-PE PE conjugated  $469.00/100T

-rabbit polyclonal

-antigen: active human caspase-3 fragment

-antibody recognizes a conformational epitope which is exposed by activation-induced cleavage or denaturation of the inactive enzyme.

-at optimal concentrations only sees active caspase-3 (approx 50 fold preference for active form compared to the inactive form). At higher concentrations may cross with inactive form.

-use: histochemistry (acetone fixed frozen sections, cytopsins), flow cytometry. In formalin fixed paraffin sections and western blot will recognize both inactive and active form. In immunoprecipitation, preferentially recognizes active caspase-3 but may also see native form at high concentrations.


Mouse anti-CPP32/caspase-3  (Human)

cat #RDI-CPP32abm         $438.00/50ug

      #RDI-CPP32abm-1     $750.00/150ug

       RDI-CPP32abm-RP $500.00/vial 50ug  (HRP conjugated)

      RDI-CPP32abm-RPX  $812.00/vial 150ug (HRP conjugated)

-suitable for use on western blot (approx 0.5ug/ml), immunoprecipitation (native or denatured conditions), reactive with human .Blots at 32Kda.

-antigen: fragment from human CPP32 1-219 (p20 region)

-mouse IgG2a, packaged as 50ug (200ul) or 150ug (600ul) (at 0.25mg/ml in 50% glycerol, PBS + 1.5mM NaN3 & 1mg/ml BSA)

For In Vitro Research Use Only


Mouse anti-CPP32 (Caspase-3) Monoclonal (Mouse/Rat)

cat# RDI-CPP32abm-46           $438.00/vial 50ug

       RDI-CPP32abm-46X         $750.00/vial 150ug

clone ref# 46

isotype: mIgG1

package: 50ug (200ul) or 150ug (600ul) (at 0.25mg/ml in 50% glycerol, PBS + 1.5mM NaN3 & 1mg/ml BSA)

antigen: a protein fragment corresponding to amino acids 25-145 from mouse Caspase-3.

Use: Suitable for western blot on mouse (rat expected but not verified) using approx 0.25-0.5ug/ml on   total cell lysate of RSV-3T3 (positive control).   Major band =Mw 32 kDa. Also suitable for   immunofluorescence (tested on RSV-3T3 cells).

Storage: Store at -20 DEG C upon receipt. . Recommend aliquoting. Avoid frequent warming cycles.

-copyright by owner

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

cat#RDI-RSV3T3/CPX $250.00/500ug vial


Rat Caspase 3 (Recombinant)

cat#RDI-TRK8C1   $688.00/5ug vial  $625.00/vial 2+ Bulk on request

Application: Caspase-3 (also known as CPP32, Yama, or Apopain) is a member of the cysteine proteases family. Similar as other caspases, caspase-3 also exists in cells as an inactive proenzyme. During the initiation of apoptosis the proenzyme is processed at aspartate residues to form active caspase-3. Involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a 216-asp-|-gly-217 bond. Cleaves and activates sterol regulatory element binding proteins (SREBPS) between the basic helix-loop- helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Cleaves DFF45 and thus activates DFF40.

In combination with caspase 3 activity assays, the active recombinant caspase 3 is useful in biological screening of caspase substrates and inhibitors. The recombinant enzyme can also be used as a positive control in caspase 3 assays.

Source: The rat Caspase 3 cDNA was synthesized using mRNA from rat brain. The purified active recombinant rat caspase 3 with C-terminal 6xHis Tag was expressed in E. coli. Heterodimer of a 17 kDa (p17) and a 12 kDa (p12) subunit.

Presentation: µg of protein in 200µl of storage buffer containing 50% glycerol.

Liquid at -20ºC.

Approximately, 180,000 Units of activity per 1mg of protein. One unit of the recombinant caspase 3 is the enzyme activity that cleaves 1 nmol of the caspase substrate z-DEVD-AMC (AMC: aminomethylcoumarin) per hour at room temperature (+20ºC) at 20 µM substrate concentration

Analysis: Purity more 98% by SDS-PAGE.Immunological activity confirmed by reaction with monoclonal antibody that is specific for the rat caspase-3 in immunoblotting.

The protein concentration was determined using the Bradford reagent.

Storage: At -20 0 C in liquid form. For long storage -70ºC is recommended.


Mouse anti-CPP32 (Caspase-3) clone JMH62 (paraffin)

cat# RDI-CPP32abm-62   $594.00/vial

Package: 1ml lyophilized tissue culture supernatant containing 15mM sodium azide. Reconstitute with 1ml of sterile water , let set at least 30 minutes 4 DEG C, Mx well before using.

Clone: JMH62

Isotype: mIgG1

Immunogen: Prokaryotic recombinant protein corresponding to a 182 amino acid fragment of the CPP32 molecule (caspase-3).

Hybridoma: Mouse myeloma (p3-NS1-Ag4-1).

Reactivity: Cysteine protease protein(CPP)-32. a member of the interleukin-1B-converting enzyme (ICE) family of mammalian proteases.

Use: Effective on paraffin was embedded tissue using the EDTA pH8.0 high temperature antigen unmasking technique:

(not recommended for frozen sections)

Typical working dilution 1:25-1:50.

High temperature antigen unmasking using technique 60 minutes primary antibody incubation at 25"C. Standard ABC technique.

Western blotting: typical working dilution: 1:100

Positive controls: Tonsil

Staining pattern: Cytoplasmic

Storage: Store unopened lyophilized antibody at 1'C. Under these conditions, there is no significant loss in product performance up to the expiry date indicated on the vial label The reconstituted antibody is stable for at lease two months when store at 4'C. For long term storage, it is recommended that aliquots of the antibody are frozen at -20'C (frost-free freezers are not recommended). Repeated freezing and thawing must be avoided. Prepare working dilutions on the day of use.

FOR RESEARCH USE ONLY-Not for use in diagnostic procedures

Background:

Cysteine protease protein (CPP)-32 is a member of the interleukin-1 beta-converting enzyme (ICE) family of mammalian proteases which specifically cleaves substrates at the C-terminal side of aspartic acid residues. Members of this family have been implicated in apoptosis and CPP32 (caspase-3) is thought to act as a control mediator of programmed cell death in mammalian cells. CPP32 is synthesised as an inactive 32kD proenzyme and is processed during apoptosis to its active form which is responsible for the cleavage of poly (ADP-ribose) polymerase (PARP), actin and sterol regulatory element binding protein  (SREBP) during apoptosis. CPP32 is detected immunohistochemically in epithelial cells of skin, renal proximal tubules and collecting ducts, epilhmorlicular cells of the thymus and bronchial, colonic and salivary duel epithelia. Chondrocytes, bone osteocytes, megakaryocyles, mature  neutrophils of bone marrow and plasma cells of the tonsil, lymph node and bone marrow also stain intensely, This antibody stains the majority of intermediate to high grade non- Hodgkin's B cell lymphomas, mantle cell lymphomas,  Burkiti's lymphomas and immunoblastic lympohomas.

References:

Krajewska M. Wang H-G. Krajewski S. et al., Immunohistochemical analaysis of in vivo patterns of expression of CPP32 (Caspase-3),a cell death protease. Cancer Research 57: 1605-1613 (1997).

Nicholson D.W. Ambereen A. Thomberry NA. et al, Identification and inhibition of the ICE-CED-3 protease necessary for mammalian apoptosis, Nature 376:37-43 (1995).

Fernandes-Alhemri T, Lrtwack g and Alriemri E S CPP32, a novel human apoptotic protein with homology to Casenorhehdotos elegans. cell death protein Ccd-3 end mammalian interleukin-1B converting enzyme. The Journal of Biological chemistry 269(49)-30761-30764 (1994).


-See also recombinant active caspase-3 cat#RDI-CASPASE3-AG  $500.00/5ug  $750.00/10ug

DATA SHEET: mouse anti-human CD95 clone DX2  (activation use)

no azide formulation:  cat#RDI-CD95-DX21X    $688.00/0.5mg

Prep: purified from tissue culture supernatant via Protein   G affinity chromatography.

Species: mouse IgG1k

CLONE: DX2

Activity: Reacts with the 45kD transmembrane cell surface FAS or APO-1 antigen, designated CD95. CD95 is expressed on a variety of normal and neoplastic cells, and is a polypeptide which plays a role in the programmed sequence of events leading to cell death (apoptosis). the DX2 clone specifically reacts with murine L cells, murine L1210 leukemia cells and murine P815 mastocytoma cells transfected with human Fas cDNA but not with untransfected parental cell lines. Cross linking with DX2 delivers an apoptotic signal indicating that DX2 recognizes a functional epitope of CD95 antigen.

Application: -Histochemistry for staining acetone fixed, frozen tissue sections.

-flow cytometry-Titer for each application

References:

1) Schlossman, S. et al, ed.s 1995, Leucocyte Typing V, White Cell Differentiation Antigens, Oxford University Press, NY.

2) J. Exp. Med, 177:1547

3) Cell 66:233

Storage: Store at 4 DEG C.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or   for diagnostics.


Rabbit Anti-Caspase-3-Active Form (CPP32) MONOCLONAL

cat# RDI-CASP3ACTabRm     $406.00/vial

Package: 25ug in 0.050ml PBS pH7.2 with 0.09% sodium azide

antigen: active human caspase-3 fragment

Clone: C92-605

Specificity: The C92-605 monoclonal antibody (mAb) specifically recognizes the active form of caspase-3 in human and mouse cells It does not recognize the proenzyme form in any assay so far tested. The mAb was originally characterized on human and mouse cells by immunohistochemical staining of apoptotic and non-apoptotic populations and shown to only recognize cells or tissues induced to undergo apoptosis.

Use: -flow cytometry: approx 0.125-0.5ug/million cells ,using Jurkat T cells treated with 4uM camptothecin for 5 hours to induce apoptosis,cells were then fixed and permeabilzied and stained with approx 0.25ug/million cells for 20 min at room temp in dark. Cells were then washed permeabilzation buffer, stained with goat anti-rabbit FITC cat#RDI-711096152. approx 50% of treated cels were positve for caspase-3 staining.

-histochemistry: acetone fixed frozen sections approx 0.25-1ug/ml. controsl include untreated Jurkat T cells and treated cells with 6uM camptothecin for 4 hours, ABC/Dab detection -for mice, untreated or treated with 100ug anti-Fas Mab (cat#RDI-MCD95-XP $625.00/0.5mg, clone J02) injected i.p. to induce apoptosis, sacrificed after 6 hours. Cytospins of JurKat cells or frozen tissue sections of mouse liver were acetone fixed and stained with antibody followed by ABC/Dab detection.

-immunoprecipitation: approx 1ug/5 million Jurkat T cells, must titer to optimize which form is Immunoprecipitated). (treated cells with 6uM camptothecin for 5 hours to induce apoptosis.

Background: The caspase family of cysteine proteases plays a key role in apoptosis and inflammation (reviewed in 1). Caspase-3 (CPP32, Yama, apopain) is a key protease that is activated during the early stages of apoptosis and, like other members of the caspase family, is synthesized as an in- active proenzyme that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another protease. The processed forms of caspases consist of large (17-22 kD) and small (10-12kD) subunits which associate to form an active enzyme. Active caspase-3, a marker for cells undergoing apoptosis, consists of a heterodimer of 17 and 12 kD subunits which is derived from the 32 kD proenzyme.2 Active caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the cytoplasm, e.g.,D4-GDI and Bcl-2(3). and in the nucleus, e.g.   PARP.

ref:

For Research Use Only


Active Recombinant Human Caspase-3 (CPP32)

CAT#: RDI-CASPASE3-AG   $500.00/5ug

            RDI-CASPASE3-AG1  $750.00/10ug

Packaging: vial of 5ug enzyme in 25 ul in 50 mM Tris, pH 8.0,with 100 mM NaC1, 50 mM imidazole.

Source: E.coli.(When expressed in E. coli, caspase-3, spontaneously undergoes autoprocessing to yield the subunits characteristic of the active enzyme).

Activity: The rate of caspase enzymatic hydrolysis was measured by release of AMC from the Ac-DEVD-AMC caspase substrate as emission at 440 nm upon excitation at 380 nm using a spectrofluorometer.

USAGE: The active enzyme is designed to be used in caspase assays.

Storage: Store the enzyme at -80'C. The thawed active enzyme is stable at 4'C for at least a week. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes, which can greatly alter product stability.

BACKGROUND

Caspases are cysteine proteases that play a centrl role in apoptosis (reviewed in 1 and 2). The caspace family was

discovered by searching human cDNA libraries for sequences homologous to ced-3, aC. elegans death gene that is required for normal apoptosis during development. The first mammalian homolog of ced-3 to be identified was ICE(interleukin-1B-converting enzyme). Subsequent numerous human ced-3 homologues were repidly identified which led to multiple names for many of the molecules. To achieve consistency,"caspase" was adopted as a root name for all family members. The name was selected based on two catalytic properties of these enzymes, the "c" reflects a cysteine protease mechanism and "aspase" refers to their unique ability to cleave after aspartic acid. There are at least 10 members, caspase- 1(ICE), caspace-2 (ICH-1), caspase-3 (CPP32), Yama, apopain), caspase-4 (TX-ICH-2, ICErel-II), caspase-5 (ICErel-III), caspase- 6 (Mch2), caspase-7(Mch3, ICE-LAP3, CMH-1), caspase-8 (MACH,FLICE, Mch5), caspase-9 (ICE-LAP6, Mch6), and caspase- 10(Mch4). Each caspase is synthesized as an inactive proenzyme that is processed by cleavage at asparte residues by another protease or by self-proteolysis. The processed forms consist of large (17-22 kD) and small (10-12 kD) subunits which assocate to  form an active enzyme. The activation of some of these caspases has been shown to occur during apoptosis.

Caspase-3,-6,-7, and -8 have been whown to play a role in apoptosis induced by the death receptors Fas and tumor necrosis factor receptor type 1 (TNFR1).One of their substrates is poly (ADP ribose) polynrase (PARP). PARP is an enzyme that is involved in DNA repair and genomic maintenance. Activated caspase 3, 6,7 and 8 can all clleave PARP from its 116 kD form to an 85 kD residual fragment. The cleavage separates the DNA-binding domain in the N-terminus of PARP from its C-terminus catalytic domain, and results in loss of normal PARP function. The cleavage site in PARP is C-terminal to Asp-216. The upstream sequence of the PARP cleavage site, DEVD (Asp-Glu-Val-Asp), is utilized as a basis for highly specific caspase-3 substrates such as Ac(N-acetyl)- DEVD-AFC (7-amino-4-trifluoromethylcourmarin)and Ac(N-acetyl)- DEVD-AMC (7-amino-4-methylcoumarin) as well as the caspase-3 aldehyde inhibitor Ac-DEVD-CHO.

For In Vitro Research Use Only

see also antigens for other caspases (caspase-6, caspase-7, caspase-8)


Return  (alphabetical antibody .index)

RDI Divison of Fitzgerald Industries Intl

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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