rev: September 19, 1999

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ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of researchd 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.

MOUSE MONOCLONAL  ANTIBODY TO: 5'-BROMO-2'DEOXYURIDINE

PRODUCT CODE   RDI-PRO61015    $375.00/vial

Also available FITC labeled cat#RDI-PRO61041   $375.00/vial 100T

   FITC conjugated with evans blue cat#RDI-PRO61016  $375.00/vial 100T)

CLONE  BU5.1

ISOTYPE  IgG2a

IMMUNIZING ANTIGEN   Bromodeoxyuridine (BrdU), no cross-reaction with thymidine

SOURCE  Mouse ascitic fluid

PURIFICATION METHOD  Protein A affinity chromatography

SPECIFICITY    BrdU incorporation during in vitro growth has been found in all systems tested so far: mammalian cell lines, e.g. P3X-63Ag8, McCoy, MCF-7; insect cell lines, e.g. Cp 169; bacterial plasmids(after incorporation of BRdu).

-excellent for in situ hybridization studies

APPLICATIONS

*This antibody is a valuable tool for the detection of S-phase (DNA-synthesizing) cells. Using the BrdU monoclonal antibody detection system the radioactive hazards and waste problems encountered with 3H-thymidine labelling techniques can be avoided

*Immunodot assays

*In situ hybridisation

Antigen recognized: after incorporation of Brdu, all DNA containing species

REFERENCES

Jirikowski, G. F. et al. Histochem. 91, 51-53 (1989)

Hosihino, T. et al. Cytometry 6, 627-32 (1985)

Jirikowski, G.F. et al, Molecular and Cellular Probes 2, 59-64 (1988)

PRESENTATION The monoclonal is presented as 100µg of lyophilysed material. Reconstitute with 1ml distilled water. We recommend that each laboratory determine an optimum working titre for use in its particular application.

-when reconstituted with 1ml distilled water, dilute further 1:10

STORAGE For use within 1 month of purchase store at +4oC, for long term storage aliquot antibody into small volumes and store at -20oC.

-see also new sheep polyclonal anti-brdu

Staining of BrdU-labelled DNA in Proliferating Cells with MAB Bu5.1

Incorporation of 5'-Bromo-2'Desoxyuridin (BrdU) into DNA 5'-bromo-2'-desoxyuridin (BrdU) is incorporated into the DNA of S-Phase (DNA-synthesizing) cells.

With Mab Bu 5.1 the proportion of cells in the S-phase of the cell cycle can be easily identified because this Mab is specific for Brdu-substituted DNA.

BrdU is added to the culture medium at a final concentration of 10-20uM together with 2'-desoxycytidine (20-50uM).

For routine work, pulses of 1-2 hours are recommended.

Short pulses (i.e. brief incubation of Brdu within the culture medium of 10min are detectable)

Indirect Immunofluorescence Microscopy

Monolayer cells

1) Wash cells, grown on slides or cover slides or cover slips, twice with PBS

2) Fix cells with cold 70% ethanol for 20min (at this stage, cells may be kept for 1 month at -20 DEG C)

3) Denatue DNA with 2.5N HCL for 20 min

4) Wash 3X with PBS

5) Incubate with Mab BU 5.1 for 30 minutes at room temp

6) Wash 3X with PBS

7) Add fluorochrome-conjugated second antibody (e.g. goat anti-mouse FITC conjugate) in appropriate dilution (incubate for 30min)

8) Wash 3X with PBS

9) Mount dry samples with standard mounting medium and evaluate with fluorescence microscope.

Suspension Cells:

1) Wash and spin cells twice with PBS (250g, 7min)

2) Resuspend cells in 3 vol PBS (0 DEG C) and fix cells by adding 7 vol 96% ethanol (0 DEG C) whilst mixing the cell suspension. Incubate for 20 min

3) Denature cells by adding one equal volume of 4N HCL to fixed cell suspension (20min, roomt emp)

4) Carefully wash and spin three times with PBS to remove HCL (250xg, 7min)

5) Staining of Brdu-substituted DNA is perfromed as described above under Monolayers steps 5 through 9. The washing steps may be reduced in order to minimize cell loss during centrifugation

DIRECT IMMUNOFLUORESCENCE MICROSCOPY (with FITC conjugates)

-cat#RDI-PRO61016 or RDI-PRO61041 are supplied in ready-to-use form. Cell prcessed accoriding to steps 1-4 are stained by adding 25ul of the Bu 5.1 FITC conjugate. After 30 minute incubation, the cells are washed twice, embedded and are then ready fro examination.

RDI Divison of researchd Industries Intl

San Jose, 95123 CA Snell ave 658

USA

or 408-780-0908

EMAIL:margaret@cellular-research.com

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