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					                                      IMMUNO-SPIN TRAPPING

   DMPO, pAb                                                           ALX-210-530-R050                                                              50 µl

Introduction
Detection of protein-derived radicals in isolated proteins has               Protein + Cu(II) + H2O2
typically required the utilization of direct electron spin resonance
(ESR) and/or spin trapping ESR. ESR can be applied both to
understand the mechanisms of protein radical formation and
for structural identification of the amino acid-derived radical in-         Protein-centered Radicals
                                                                                                                                 ESR (EPR)
volved. However, ESR measurements require significant levels                     (t 1/2 =µsec-sec)
of protein and of its derived radicals (greater than micromolar)                             + DMPO
and rely on rather specialized and costly equipment. Moreover,
detection of protein radicals by ESR becomes a difficult task in          DMPO-protein Radical Adducts
                                                                                                                                 ESR-spin Trapping
cells and tissues.                                                             (t 1/2 =sec-min)
Immuno-spin trapping is based on the fact that certain amino                              - e-
                                                                                          - H+
acid-derived radicals (most notably, but not exclusively, tyrosyl
radicals) can react with the spin trap 5,5-dimethyl-1-pyrroline-N-       DMPO-protein Radical-derived
                                                                               Nitrone Adducts                                   Immuno-spin Trapping
oxide (DMPO) to form a protein-DMPO nitroxide radical adduct,                (t 1/2 =days-months)
which can then be oxidized by one electron to the corresponding
ESR-silent protein-DMPO nitrone adduct. Indeed, DMPO spin                FIGURE: Schematic overview of approaches to detect protein radicals.
adducts are not stable, have a half-life on the order of minutes,
and tend to decay under oxidizing conditions to the rather stable
DMPO nitrone. Rabbit polyclonal anti-DMPO nitrone antiserum
                                                                       P. Mason: Immuno-spin trapping of DNA radicals; Nat. Methods
(Prod. No. ALX-210-530) has been developed and validated in
                                                                       3, 127 (2006)), which broadens even more the scope of applica-
Mason’s laboratory (C. D. Detweiler, L. J. Deterding, K. B. Tomer,
                                                                       tions of this breakthrough technique in free radical biology and
C. F. Chignell, D. Germolec, R. P. Mason: Immunological identifi-
                                                                       medicine.
cation of the heart myoglobin radical formed by hydrogen perox-
ide; Free Radic. Biol. Med. 33, 364 (2002)) and a series of studies    Adapted from: Immuno-spin trapping: A breakthrough for the
have shown its utility to demonstrate protein radical formation in     sensitive detection of protein-derived radicals, a commentary on
different proteins exposed to a variety of oxidants.                   “Protein radical formation on thyroid peroxidase during turnover”;
In summary, immuno-spin trapping is proving to be a potent,            R. Radi; Free Radic. Biol. Med. 41, 416 (2006).
sensitive, and accessible method to detect low levels (e.g. great-
er than nanomolar) of protein-derived radicals produced in vitro
and potentially, and yet to be established, in vivo. Moreover, it       Selected Review Article
has just been established that anti-DMPO nitrone antibodies can          Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time
                                                                         and space with immuno-spin trapping: R. P. Mason; Free Radic. Biol. Med. 36, 1214
be utilized for the detection of DNA-derived radicals in the form        (2004)
of DNA-DMPO nitrone adducts (D. C. Ramirez, S. E. Mejiba, R.




                                                                                           incorporating
Product Flyer
International Edition 1-Nov-2009                                                          www.enzolifesciences.com
DMPO, pAb
ALX-210-530-R050                                                                                                                                                                                                                                                                                      50 µl
From rabbit. IMMUNOGEN: DMPO-octanoic acid conjugated to
ovalbumin. SPECIFICITY: Recognizes DMPO (Prod. No. ALX-
430-090), DMPO-octanoic acid and DMPO-protein adducts.
Does not cross react with non-adducted proteins. APPLICA-
TION: ELISA, IHC, WB.
Widely Published                                                                                                                                                                                                                                                                                                                                                                         Switzerland & Rest of Europe
LIT: Immunological identification of the heart myoglobin radical formed by hydro-                                                                                                                                                                                                                                                                                                        ENZO LIFE SCIENCES AG
gen peroxide: C. D. Detweiler, et al.; Free Radic. Biol. Med. 33, 364 (2002)  Im-                                                                                                                                                                                                                                                                                                       Industriestrasse 17, Postfach
munochemical detection of hemoglobin-derived radicals formed by reaction with
hydrogen peroxide: involvement of a protein-tyrosyl radical: D. C. Ramirez, et al.;                                                                                                                                                                                                                                                                                                      CH-4415 Lausen / Switzerland
Free Radic. Biol. Med. 34, 830 (2003)  Identification of free radicals on hemo-                                                                                                                                                                                                                                                                                                         Tel. + 41/0 61 926 89 89
globin from its self-peroxidation using mass spectrometry and immuno-spin trap-
ping: observation of a histidinyl radical: L. Deterding; J. Biol. Chem. 279, 11600                                                                                                                                                                                                                                                                                                       Fax + 41/0 61 926 89 79
(2004)  Protein radical formation during lactoperoxidase-mediated oxidation of                                                                                                                                                                                                                                                                                                          info-ch@enzolifesciences.com
the suicide substrate glutathione: immunochemical detection of a lactoperoxidase
radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct: Q. Guo, et al.; J.
Biol. Chem. 279, 13272 (2004)  Mechanism of hydrogen peroxide-induced Cu,Zn-
superoxide dismutase-centered radical formation as explored by immuno-spin                                                                                                                                                                                                                                                                                                               North/South America
trapping: the role of copper- and carbonate radical anion-mediated oxidations:
D. C. Ramirez, et al.; Free Radic. Biol. Med. 38, 201 (2005)  Identification of the                                                                                                                                                                                                                                                                                                     ENZO LIFE SCIENCES INTERNATIONAL, INC.
myoglobin tyrosyl radical by immuno-spin trapping and its dimerization: C. D. Det-                                                                                                                                                                                                                                                                                                       5120 Butler Pike
weiler, et al.; Free Radic. Biol. Med. 38, 969 (2005)  Immuno-spin trapping: detec-
tion of protein-centered radicals: D. C. Ramirez, et al.; Curr. Prot. Toxicol. 2, 17.7.1                                                                                                                                                                                                                      FIGURE: Confocal microscopy images of the colocalization of                Plymouth Meeting, PA 19462-1202
(2005)  Superoxide generation from mitochondrial NADH dehydrogenase induces                                                                                                                                                                                                                                  catalase (red stain) and protein-DMPO adducts (green stain)                USA
self-inactivation with specific protein radical formation: Y. R. Chen, et al.; J. Biol.                                                                                                                                                                                                                       obtained by treating mouse hepatocytes (2.5x106 cells/ml) with
Chem. 280, 37339 (2005)  Immunochemical detection of nitric oxide and nitrogen                                                                                                                                                                                                                               HOCl.                                                                      Tel. 1-800-942-0430 / (610) 941-0430
dioxide trapping of the tyrosyl radical and the resulting nitrotyrosine in sperm whale                                                                                                                                                                                                                                                                                                   Fax (610) 941-9252
myoglobin: K. Nakai and R. P. Mason; Free Radic. Biol. Med. 39, 1050 (2005)                                                                                                                                                                                                                                  Picture courtesy of Dr. M. G. Bonini, National Institute of Environ-
Immuno-spin trapping of DNA radicals: D. C. Ramirez, et al.; Nat. Methods 3, 123                                                                                                                                                                                                                              mental Health Sciences, Research Triangle Park (RTP), NC, USA.             info-usa@enzolifesciences.com
(2006)  Immuno-spin trapping of hemoglobin and myoglobin radicals derived from
nitrite-mediated oxidation: A. Keszler, et al.; Free Radic. Biol. Med. 40, 507 (2006)
 Protein radical formation on thyroid peroxidase during turnover: M. Ehrenshaft,
et al.; Free Radic. Biol. Med. 41, 422 (2006)  Protein radical formation on thyroid                                                                                                                                                                                                                                                                                                     Benelux
peroxidase during turnover: M. Ehrenshaft, et al.; Free Radic. Biol. Med. 41, 422                                                                                                                                                                                                                                                                                                        ENZO LIFE SCIENCES BVBA
(2006)  Immunolocalization of hypochlorite-induced, catalase-bound free radi-
cal formation in mouse hepatocytes: M. G. Bonini et al.; Free Rad. Biol. Med.                                                                                                                                                                                                                                                                                                            Melkerijweg 3
42, 530 (2007)  Mitochondrial Complex II in the Post-ischemic Heart: Oxida-                                                                                                                                                                                                                                                                                                             BE-2240 Zandhoven / Belgium
tive injury and the role or protein S-Glutathionylation: Y. R. Chen, et al.; J. Biol.
Chem. 282, 32640 (2007)  Mitochondrial dysfunction in SOD1G93A-bearing                                                                                                                                                                                                                                                                                                                  Tel. +32/0 3 466 04 20
astrocytes promotes motor neuron degeneration: prevention by mitochondrial-                                                                                                                                                                                                                                                                                                              Fax +32/0 3 466 04 29
targeted antioxidants: P. Cassina, et al.; J. Neurosci. 28, 4115 (2008)  Direct
evidence of iNOS-mediated in vivo free radical production and protein oxidation                                                                                                                                                                                                                                                                                                          info-be@enzolifesciences.com
in acetone-induced ketosis: K. Stadler, et al.; Am. J. Phys. Endocrin. Metab. 295,
E456 (2008)  Immuno-spin trapping of a post-translational carboxypeptidase
B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide
synthase in acute septic mice: S. Chatterjee, et al.; Free Radic. Biol. Med. 46,                                                                                                                                                                                                                                                                                                         Germany
454 (2009)  Immuno-spin trapping of protein and DNA radicals: "tagging" free
                                                                                                                                                                                                                                                                                                                                                                                         ENZO LIFE SCIENCES GmbH
radicals to locate and understand the redox process: S. E. Gomez-Mejiba, et
al.; Free Radic. Biol. Med. 46, 853 (2009)                                                                                                                                                                                                                                                                                                                                               Marie-Curie-Strasse 8
                                                                                                                                                                                                                                                                                                                                                                                         DE-79539 Lörrach / Germany
                                                                                                                                                                                                                                                                                                                                                                                         Tel. +49/0 7621 5500 526
                                                                                                                                                                                                                                                                                                              Application Note                                                           Toll Free:  0800 6649518
                                                                                                                                                                                                                                                                                                                                                                                         Fax +49/0 7621 5500 527
                                                                                                                                                                                                                                                                                                               Immuno-spin Trapping of DNA                                               info-de@enzolifesciences.com


                                                                                                                                                 SPIN P
                                                                                                                                                                                 ROBES                                                                                                                         Radicals
                                                                                                         RAPS &                                                                                                                                                                                                                                                                          UK & Ireland
                                                                        SPIN T                                                                                                                                                                                                                                 The detection of DNA radicals by immuno-
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                                                                                        Spin Tr products are quality tested by sis are our speciality
                                                                                                                                       ctrosco


                                                                                 Stable
                                                                                                                               ESR spe

                                                                                                                                                                                                                                                                                                               spin Trapping (IST) is based on the trapping                              ENZO LIFE SCIENCES (UK) LTD.
                          urity &
                                                                                             • All                           the
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                    High P spin traps for in vitro and in vivo
                                                                       tions                                                                                           tion & cus
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                                                                                                                                                                                                                                                                                                               of radicals with 5,5-dimethyl-1-pyrroline                                 Palatine House
                                                                                                                                                                                                                                 ; 2-D
                                                                                                                                            • Bulk                                                                      -N-oxide
                                                                                                                                                                                                               pyrroline1-oxide]
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                                                                                                                                                                  -119-M0                                               (Prod. No. O-, N-, S-,
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                             dependen hilic (Kp=0.16 ethyl-1-pyrroline tered radica ation Roubaud, et al.;     and                  Anal. Bioch               DMPO ethyl-1-pyrroline                                                                                  eable hy-



                                                                                                                                                                                                                                                                                                               anti-DMPO serum. IST combines the sim-
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                                    BMPO Butoxycarbonyl-5

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                                                                                                                                                                               N-                               ner; Free website.
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                                                              DEPMPO
                                                                                               BMPO
                                                                                                        (high
                                                                                                                 purity)



                                                                                                               DMPO
                                                                                                                         (high
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                                                                                                                                                                                                                 incorp
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                                                                                                                                                                                                                                                                                                               and complements existing methods for the
                                                                                                                                                                                                                                                                                                               measurement of oxidatively generated DNA                                  For Local Distributors please
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                                                                                                                                                                                                                                                                                                               damage.                                                                   visit our Website.

                                                                                                                                                                                                                                                                                                               LIT: Immuno-spin trapping of DNA radicals: D. C. Ramirez, et al.; Nat.

      REQUEST YOUR COPY                                                                                                                                                                                                                                                                                        Methods 3, 123 (2006)  Immuno-spin trapping analyses of DNA radi-
                                                                                                                                                                                                                                                                                                               cals: D. C. Ramirez, et al.; Nat. Protoc. 2, 512 (2007)  Immuno-spin
                                                                                                                                                                                                                                                                                                               trapping of protein and DNA radicals: "tagging" free radicals to locate
     OF THE LATEST EDITION!                                                                                                                                                                                                                                                                                    and understand the redox process: S. E. Gomez-Mejiba, et al.; Free
                                                                                                                                                                                                                                                                                                               Radic. Biol. Med. 46, 853 (2009)
                                                                                                                                                                                                                                                                                                                                                                                                         incorporating



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