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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.


Anti-Alzheimer precursor protein A4

-Monoclonal antibody to the Alzheimer precursor protein A4 from man from mouse-mouse-hybrid cells (clone 22C11) lyophilized immunoglobulin, stabilized

   cat#RDI-ALZHPA4abm    $531.00/50ug vial

Product Description:

Preparation: To obtain monoclonal antibodies Balb/c mice were immunized with purified recombinant Alzheimer precursor A4 (pre-A4 (695)) fusion protein. Spleen cells were hybridized with myeloma cells of the cell line Sp 2/0 Ag 14. The antibodies have been purified from ascites fluid by ion exchange chromatography and diluted in 10 mM potassium phosphate buffer, 50 mM NaC1, 5 mg/ml bovine serum albumin (BSA),0.1% Kathon(tm) (w/v); pH 7.5 and lyophilized.

The antibody belongs to the IgG1 subclass.

Dissolving the lyophilisate in 1 ml redist. water results in a concentration of 50 ug antibody/ml.

Recommended working concentration: 5-10 ug/ml.

Stability: The lyophilized antibody is stable when stored at 4'C. The reconstituted antibody solution is stable for one year when stored at 4'C. The solution can be stored in aliquots at -20'C.Repeated freezing and thawing should be avoided.

Specificity: The antibody reacts with pre-A4 from man. Cross-reactivity was shown with fish,rat, muse and monkey. The antibody recognizes a N-terminal epitope on the pre-A4 molecule.

Application:

The antibody is suitable for immunohistochemical detection of pre-A4 molecules on sections, and for Western Blots.


Working instruction for peroxidase-anti-peroxidase reaction

Assay principle: In the first incubation step the anti-pre-A4 antibody binds to the pre-A4 molecules of the spinal cord of rat. After washing to remove excess antibody anti-mouse Ig from sheep is used as bridging antibody. Afterwards the preparation is incubated in peroxidase-anti-peroxidase solution. The reaction is performed using the high sensitive diamino-benzidine as substrate

Required reagents:

Diaminobenzidine, A.R.

Anti-mouse Ig

Heparin

Sodium chloride,A.R.

Peroxidase-anti-peroxidase complex

Paraformaldehyde

Picric acid, A.R.

Bovine serum albumin

Normal sheep serum

Sucrose

Tris-HC1

Triton X-100

Hydrogen peroxide

Preparation of the solution

I. Buffer

A. 100 mM sodium phosphate; pH 7.2.

B. 100 mM Tris-HC1,150mM NaC1; pH 7.4.

II. Heparin solution

0.05% Heparin (w/v) in buffer A.

III. Paraformaldehyde-picric acid solution

4% Paraformaldehyde (w/v); saturated 15% picric acid solution

(v/v) in buffer A.

IV.Buffer for anti-pre-A4 antibodies

10% normal sheep serum (v/v); 2% bovine serum albumin (w/v);

0.1 - 0.5% Triton X-100 (v/v) in buffer B.

V. Anti-pre-A4 solution

Dilute reconstituted antibody solution 1:5 - 1:10 with solu-

tion IV (5-10 ug antibody/ml).

VI.Washing buffer

Buffer B.


VII. Anti-mouse immunoglobulin solution

Anti-mouse Ig in buffer B with 2% bovine serum albumin (w/v)

5% normal sheep serum (v/v).

VII. Peroxidase-anti-peroxidase solution

Peroxidase-anti-peroxidase in buffer B with 2% bovine serum

albumin (w/v); 5% normal sheep serum (v/v).

IX. Substrate solution

Diaminobenzidine, 1mg/ml; 0.01% H2O2 (w/v) in buffer A.

Stability of the solutions:

Solutions I (A and B) are stable for 1 week at 4'C.

Solutions II and III must be prepared immediately before use.

Solutions IV and V are stable for 4 weeks at 4'C.

Solutions VI, VII and VIII are stable for 1 week at 4'C.

Solution IX must be freshly prepared each day.

Procedure:

Fixation:

Spinal cord tissue from above mentioned species [transcardial perfusion, modification of the method described by Houser et al.] The animals are first transcardially perfused with heparin solution (II) saturated with oxygen (100-200 ml for rats) and then with the paraformaldehyde-picric acid solution (III). 400 ml of  solution III is required for rats. The tissue is prepared immediately and then given 1 h postfixation in the same solution (III). From the tissue vibratome or cryo sections are cut (ca. 50 um).

Procedure:

Incubation of the sections is carried out by flotation in suitable small vessels. They are mounted on microscope slide after the assay procedure.

Blocking of endogenous peroxidase with 0.5% H2O2 (w/v).

Wash sections 3 times in buffer B.

Incubate sections in antibody solution V overnight at 4'C.

Wash sections 3 times for 5 min in buffer B.

Incubate sections in solution VII for 1 hr. at room temperature.

Wash sections 3 times for 5 min in buffer B.

Incubate sections in solution VIII for 1 hr at room temperature.

Wash sections 3 times for 5 min in buffer B.

Incubate sections in substrate solutions IX at room temperature until a reddish-brown coloration is clearly visible. A parallel negative control must remain unstained during this incubation period.

Wash the sections in buffer B; mount on microscope slide coated with chrome alum gelatine, allow to dry, dehydrate and cover.

Test Procedure:

The sensitivity and speed of the test can be adapted to the user's particular requirement by varying the quantities used, the incubation times and the temperature.

References:

1. Weidemann, A. et al. (1989) Cell 57, 115-126.

2. Koo,E.H. et al.(1990) Proc.Natl.Acad.Sci. USA, 87,1561-1565.

3. Martin,L.J. (1990) Neuron in press.

4. Houser,C.R. et al. (1983) Brain Research,266,97-119.

5. Martin,L.J. et al. (1991) Proc.Natl.Acad.Sci. USA 88,1461-1465.

6.Momming,V. et al. (1990) FEBS, 277, 261-266.

FOR 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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