Clone #

:

5F6

Antigen

:

ACR-modified keyhole-lympet hemocyanine

Form

:

Frozen (100 micro g/mL antibody in 10mM PBS containing 0.1 %NaN3 and 0.5% BSA). Purified by Protein-A.

Application

:

Immunohistochemistry.
Recommended antibody concentration is 0.5-1.0 micro gram/mL on paraformaldehyde fixed tissue.

Specificity

Specific for ACR-modified protein (especially FDP-lysine type derivative)

Subclass

Mouse IgG1,kappa

Storage

Less than -20°C

Paraffin sections were deparaffinized and rehydrated.

Sections were quenched for several minutes with 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.

Sections were incubated overnight at 4°C with a antibody at a dilution of 1 micro gram/mL.

Antibody binding was visualized by the avidin-biotin-immuno peroxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.

3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.

Hematoxylin or Eosin was used as the counter stain.

Sections from which the primary antibodies were omitted served
as negative reaction controls.

For frozen materials, samples were fixed in 10% formalin, immersed
in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan),
and stored at 80°C.

For paraffin embedded materials, samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and stored at room temperature.

Microwave treatment improves the target staining, whereas it declines
non-specific background.

Protease K treatment declines the target staining.

1)

Protein-bound acrolein: Potential markers for oxidativestress. K.Uchida, M.Kanematsu, K.Sakai, T.Matsuda, N.Hattori,Y.Mizuno, D.Suzuki,T.Miyata, N.Noguchi, E.Niki,T.Osawa
Proc.Natl.Acad.Sci.USA,95,4882-4887(1998)

2)

Protein-bound acrolein: A novel markers of oxidativestress in
Alzheimer's Disease. Noel Y. Calingasan, Koji Uchida,
and Gary E.Gibson
Journal of Neurochemistry.72(2),751-756(1999)

3)

A 1-Hour Enzyme-Linked Immunosorbent Assay for Quantitation
of Acrolein- and Hydroxynonenal- Modified Proteins by Epitope-Bound
CaseinMatrix Method. K.Satoh,S.Yamada,Y.Koike,Y.Igarashi,S.Toyokuni,T.Kumano,T.Takahata,
S.Tsuchida and K.Uchida
Analytical Biochemistry.270,323-328(1999)

4)

Red blood cells inhibit activation-induced cell death and oxidative
stress in human peripheral blood T lymphocytes. Fonseca AM, Porto G,
Uchida K, Arosa FA.
Blood. 97(10), p3152-60 (2001)

5)

Thiolation of protein-bound carcinogenic aldehyde.
An electrophilic acrolein-lysine adduct that covalently binds to thiols.
Furuhata A, Nakamura M, Osawa T, Uchida K.
J Biol Chem. 277(31), p27919-27926 (2002)

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