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ANTIBODIES  

(anti-Human and others as indicated)

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, histochemistry, 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.


Monoclonal antibody against the cytoplasmatic carboxyl fragment APP 643-695/Jonas

cat#RDI-ALZHPJabm    $500.00/100ug vial

From mouse-mouse hubrid cells (clone 2.F2.19B4), IgG1 Lyophilized immunoglobulin, stabilized.

Introduction:

One of the pathological characteristics of Morbus Alzheimer is the extracellular deposit of amyloid (B-A4-protein) in the brain Amyloid is a protealytic cleavage product of Alzheimer precursor protein (APP). APP has a receptor-like structure, is anchored in the cell membrane of neurones and possesses a strongly conserved cytoplasmatic domain . The biological function of the receptor is unknown.

APP has a molecular weight of 120 kD and is processed in an endosomal-lysosomal pathway in fragments of 8-12 kD. These fragments possess the potential amyloidogenic form of the B-44 molecule (4 kD).

Product Description:

Preparation: To obtain the monoclonal antibody, Balb/c mice were immunized with the carboxy fragment APP 643-695/Jonas. The spleen cells were then fused with myoloma cell line P3-X63-Ag8.653. The antibody thus prepared were purified by ion exchange chromatography, dissolved in 10 mM potassium phosphate: 50 mM NaC1; 5 mg/ml raffinose; Kathon CG. 0.1% (w/v); pH 7.4 and subsequently lyophilized.

Dissolving the lyophilisate in 1 ml of redist. water yields an antibody concentration of 100ug/ml.

The antibody belongs to mouse immunoglobulin subclass IgG1.

Recommended working concentration: for immunohistochemistry on paraffin sections: 10-20 ug/ml for Western blot: 10 ug/ml.

Stability: The lyophilisate is stable if stored at 4'C. The reconstituted solution is stable at -20'C. Repeated freezing and thawing should be avoided. We recommend storing the solution in suitably sized aliquots at -20'C.

Specificity: The antibody reacts with intact full-length Alzheimer precursor protein (APP) and selectively with the cytoplasmatic carboxyl fragment of APP 643-695 from humans and rats.

Application:

The antibody can be used for the immunohistochemical detection of APP and its fragments in cryo- and paraffin sections and for detection in Western Blot analysis.

Working procedure for immunohistochemical staining:

The following procedure has been developed for the detection of  APP in paraffin sections of Alzheimer tissue.

1. Prepare paraformaldehyde-fixed paraffin sections. Wash twice for 5 min. in xylene to deparaffinate. Wash the sections for 5 min. in a descending series of alcohol solutions (100%, 96%, 90%, 80%, 70%, 50%, 30%).

2. Wash the sections 3 times in deionized water.

3. Wash in TBS (50 mM Tris-HC1, pH 7.6). If peroxidase staining is to be carried out, wash the sections for 5 min. in methanol containing H2O2. 0.6% (v/v) and horse serum. 10% (v/v); this  blocks endogenous peroxidase.

4. Wash the sections for 5 min in TBS.

5. Cover the sections with 200 ul anti-Alzheimer Precursor Protein 643-695/ Jonas antibody (10-20 ug antibody/ml TBS containing horse serum, 10% (v/v) and incubate overnight or for 2 hr at 37'C in a humid chamber.

6. Wash the sections 3 times for 5 min in TBS.

7. Cover the sections with an adequate amount of anti-mouse Ig-POD solution and incubate for 1 hr. at room temperature in a humid chamber.

8. Peroxidase staining: Coat the sections with a suitable substrate solution (see below) and incubate at room temperature until a clearly visible red-brown color develops. A negative control run in parallel should remain unstained. Remove the substrate solution by washing in TBS and counterstain with hematoxylin or hemalum. Remove this staining solution by washing in TBS. Embed the sections and examine under a microscope.

Substrate solutions:

Amioethylcarbazole: Dissolve 2 mg 3-amino-9-ethylcarbazole in 1.2 ml dimethyl sulfoxide and add 28.8 ml 0.05 mM Tris-HC1. pH 7.3 and 20 ul H2O2, 30% (v/v). Prepare fresh solution daily.

Diaminobenzidine: Dissolve 25 mg 3.3'-diaminobenzidine tetrahydrochloride in 50 ml 0.05 mM Tris-HC1, pH 7.3 and add 50 ul H2O2, 30% (v/v). Prepare a fresh solution daily.

Note: The sections should not be allowed to dry up during the procedure.

Working procedure for Western Blot:

Using Western Blot analysis on extract of Alzheimer brain tissue intact full-length APP and its fragments can be detected with anti-Alzheimer precursor protein 643-695/Jonas.

Solublize tissue or cells with extraction buffer (1 ml/10% cells, 2-3 ml/g tissue, see below for buffer composition) and separate non-soluble components by centrifugation at 10,000 g. The lysate is stable if stored at -20'C and can be used directly or diluted 4x with sample buffer and boiled for 2-5 min. prior to SDS-poly-acrylamide gel electrophoresis. Transfer to nitrocellulose according to the method of Burnette (9).

Extraction buffer:

50 mM Tris, pH 7.6

2 mM mercaptoethanol

2 mM EDTA

1 mM Pefabloc

150 mM NaC1

Triton X 100', 1%

1. Dilute Alzheimer tissue extract with SDS sample buffer. Apply ca. 50 ug protein to each of the pocket of the SDS polyacrylamide gel. We recommend using an SDS polyacrylamide gradient gel (10 - 20%) under reducing conditions.

2. On completion of SDS-PAGE, transfer the separated proteins onto a nitrocellulose membrane using the Western blot technique.

3. Block unspecific binding sites by incubating the membrane in TBS + Tween 20, 0.1% and dry milk, 1%. Incubate the membrane subsequently for 10 min. on a plate shaker.

4. Incubate the membrane overnight at 4'C. with an adequate quantity of anti-Alzheimer precursor protein 643-695/Jonas ( 10 ug/ml). Alternatively, incubation can be carried out for 2 hr. at room temperature.

5. Wash the membrane 3 times for 10 min. in TBS + Tween 20,0.1%.

6. Incubate the membrane on a plate shaker for 1 hr at 37'C with an adequate quantity of suitably diluted anti-mouse Ig' as bridging antibody.

7. Incubate the membrane for 1 hr at 37'C with peroxidase-anti-peroxidase solution (PAP-complex , 1000 mU/ml).

8. Wash the membrane 3 times for 10 min in TBS + Tween 20, 0.1%.

9. Visualize the bands by incubating the membrane in a suitable substrate solution (see below).

Substrate solutions:

DAB (diaminobenzidine ( 3-4, 3-4-tetraaminobiphenyl): Dissolve 139 mM DAB, H2O2, 0.1% (v/v); in 50 mM Tris-HCI; pH 7.3. The resulting product is brown and is insoluble in water and ethanol.

CN (4-chloro-1-napthol):

Dissolve 5.6 mM CN: H2O2, 0.01% (v/v), in 50 mM Tris-HCl, pH 7.4; 150 mM NaCl. The resulting product is blue-black, insoluble in water but soluble in ethanol.

References:

1. Selkoe, D.J. et al. (1989) Cell 58, 611-612.

2. Kang, J. et al (1987) Nature 325, 733-766.

3. Ponte, P. et al (1988) Nature 331,525-527.

4. Tanzi, R.E., et al (1988) Nature 331,528-530.

5. Kitaguchi, N. et al (1988)Nature 331, 530-532.

6. Knops, J. et al. (1992) J. Biol.Chem. 267,16022-24.

7. Golde, T.E. et al (1992) Science 255, 728-730.

8. Haass, C., et al (1992) Nature 359,322-325.

9. Burnette, W.N. (1981) Anal. Biochem. 122, 195-203.

For Research Use OnlyFOR RESEARCH USE ONLY-


RDI Division 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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