BrdU antibody

Catalog No. ABIN125971

Product Details anti-BrdU Antibody

Target: BrdU (Bromodeoxyuridine (BrdU))

Reactivity: Please inquire

Host: Mouse

Clonality: Monoclonal

Conjugate: This BrdU antibody is un-conjugated

Application: Immunofluorescence (IF)

Purification: Protein A Affinity Chromatography

Immunogen: Bromodeoxyuridine

Clone: Bu5-1

Isotype: IgG2a

Application Details

Application Notes: Detection of bromodeoxyuridine incorporation in tissue and cell culture - see protocols onpage 2. Working dilution: 1/10. Incubation time: 30 min at RT.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: Staining of BrdU-labelled DNA in Proliferating Cells with Mab BU 5. 1Incorporation of 5'-Bromo-2'-Desoxyuridin (BrdU) into DNA5'-bromo-2'-desoxyuridin (BrdU) is incorporated into the DNA of S-Phase(DNA-synthesizing) cells. [Cat. No. ABIN125970(contains Evans Blue), Cat. No. ABIN125971]With Mab BU 5. 1 the proportion of cells in the S-Phase of the cell cycle can be easilyidentified because this Mab is specific for BrdU-substituted DNA. BrdU is added to the culture medium at a final concentration of 10 - 20 µM together with2'-desoxycytidine (20 - 50 µM). For routine work pulses of 1 - 3 h are recommended. Short pulses (i. e. brief incubation of BrdU within the culture medium of 10 min aredetectable). I. IndirectImmunofluorescence Microscopy1. Monolayer Cells1. Wash cells, grown on slides or cover slips, twice with PBS. 2. Fix cells with cold 70% ethanol for 20 min (at this stage, cells may be kept for 1 month at-20°C). 3. Denature DNA with 2. 5 N HCl for 20 min. 4. Wash 3x with PBS. 5. Incubate with Mab BU 5. 1 for 30 min at room temperature. 6. Wash 3x with PBS. 7. Add fluorochrome-conjugated second antibody (e. g. goat anti-mouse FITC conjugate) inappropriate dilution (incubate for 30 min). 8. Wash 3x with PBS. 9. Mount dry samples with standard mounting medium and evaluate with fluorescencemicroscope. 2. Suspension Cells1. Wash and spin cells twice with PBS (250 g, 7 min). 2. Resuspend cells in 3 vol PBS (0°C) and fix cells by adding 7 vol 96% ethanol (0° C) whilstmixing the cell suspension. Incubate for 20 min. 3. Denature cells by adding one equal volume of 4 N HCl to fixed cell suspension (20 min,room temperature). 4. Carefully wash and spin three times with PBS to remove HCl (250x g, 7 min). 5. Staining of BrdU-substituted DNA is performed as described above under I. 1. (steps 5-9). The washing steps may be reduced in order to minimize cell loss during centrifugation.

Restrictions: For Research Use only

Handling

Reconstitution: Restore in 1 mL dist. water

Buffer: PBS buffer, pH 7.4 containing 0.09 % Sodium Azide as preservative and 0.5 % BSA as stabilizer

Preservative: Sodium azide

Precaution of Use: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: This product is photosensitive and should be protected from light

Storage: 4 °C

Storage Comment: Prior to and following reconstitution store the antibody undiluted at 2-8 °C. DO NOT FREEZE!

Target Details for BrdU

Target: BrdU (Bromodeoxyuridine (BrdU))

Abstract: BrdU Products

Target Type: Chemical

Background: Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic nucleoside which is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues. BrdU can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), substituting for thymidine during DNA replication. Antibodies specific to BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA. Binding of the antibody requires denaturation of the DNA by heat or acid.