MAB3424
Anti-BrdU-Antikörper, Klon AH4H7-1 / 131-14871
Chemicon®, from mouse
Synonym(e):
BrdU
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UNSPSC Code:
12352203
NACRES:
NA.41
eCl@ss:
32160702
Clone:
131-14871, monoclonal, AH4H7-1, monoclonal
Technique(s):
flow cytometry: suitable, immunocytochemistry: suitable, immunohistochemistry: suitable
Application:
FACS, ICC, IHC
Citations:
52
Technischer Dienst
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Unterstützung erhaltenbiological source
mouse
Quality Segment
antibody form
purified immunoglobulin
antibody product type
primary antibodies
clone
131-14871, monoclonal, AH4H7-1, monoclonal
species reactivity (predicted by homology)
all
manufacturer/tradename
Chemicon®
technique(s)
flow cytometry: suitable, immunocytochemistry: suitable, immunohistochemistry: suitable
isotype
IgG1
shipped in
wet ice
target post-translational modification
unmodified
General description
Abhängig von der Molekülmasse des in das nachzuweisende Protein eingebundenen Bromdesoxyuridins
Bromdesoxyuridin (BrdU) ist ein Thymidin-Analogon und wird während der DNA-Synthese spezifisch in DNA integriert. Der monoklonale Anti-Bromdesoxyuridin-Antikörper wird zum Identifizieren von Zellen mit eingebundenem BrdU eingesetzt. Dieses immunologische Nachweisschema weist gegenüber der radioaktiven Thymidineinbindung zum Identifizieren von Zellen in der Replikation mehrere Vorteile auf. Markierung und Nachweis können am selben Tag durchgeführt werden, anstatt mehrere Tage zu warten, wie es bei der Autoradiografie von mit Tritium markierten Zellen erforderlich ist, und die Notwendigkeit zum Verwenden mehrerer Proben zum Erhalt der optimalen Aussetzungsdauer entfällt. Außerdem ermöglicht die Färbung mit Anti-Bromdesoxyuridin in Kombination mit einer Durchflusszytometrie die gleichzeitige Bewertung mehrerer Parameter. Der monoklonale Anti-Bromdesoxyuridin-Antikörper wurde zum Identifizieren von proliferierenden Zellen in Blut (Campana et al., 1988), Gewebe (Schutte et al., 1987; Hayashi et al., 1988), Tumoren (Hoshino et al., 1986; Morstyn et al., 1983) sowie zum Bestimmen von Plasmazellmarkierungsindizes (Greipp et al., 1985) eingesetzt.
Immunogen
Bromdesoxyuridin-Rinderserumalbumin-Konzentrat
Application
Forschungskategorie
Epigenetik & Zellkernfunktion
Epigenetik & Zellkernfunktion
Forschungsunterkategorie
Zellzyklus, DNA-Replikation & -Reparatur
Zellzyklus, DNA-Replikation & -Reparatur
Immunohistochemistry: (6 μg/ml)
Flow Cytometry: (0.2 μg/100 μl/10E6 cells) Optimal working dilutions must be determined by end user.
APPLICATIONS
Flow cytometry:The method below is based on that of M. Vanderlaan et al. (1986). Variations of this method exist in the literature, one consideration being the effect various fixation procedures have on the light-scattering properties of different cell populations. Procedure:
1. To label cells, pulse with 10 μM bromodeoxyuridine for 30 minutes. Harvest cells from culture.
2. Fix cells in 70% ethanol at +2-8°C for at least 30 min. Extract histones by resuspending cells in 1 mL chilled 0.1 M HCI containing 0.5% Triton X-100; incubate the suspension on ice for 10 minutes. Dilute acid with 5 mL distilled water and centrifuge at 200 x g for 10 min. Resuspend cells in 2 mL distilled water.
3. Denature cellular DNA by submerging the cell suspension into a boiling water bath for 10 min. Afterwards, quickly cool by placing the cell suspension in an ice slurry for several minutes. Wash cells in PBS that contains 0.5% Triton X-100.
4. Resuspend the cells (1-2 x 10 6 cells) in 100 μL of solution containing approximately 2 μg/mL anti-bromodeoxyuridine antibody diluted in PBS containing 0.1% BSA (0.2 μg/test). Incubate for 30 min at room temperature. Wash cells with PBS.
5. Resuspend cells in 100 μL of diluted goat anti-mouse IgG-FlTC Wash cells with PBS.
APPLICATIONS (Cont.)
Immunohistochemistry: Below is a procedure for staining cells that have been labeled with BrdU in vivo or in vitro. The procedure is based on the methods of B. Schutte et al. (1987) and D. Campana et al. (1988).
Preparation of tissue:
Inject animal with 50 mg BrdU/kg body weight. Sacrifice animal one hour later and remove organ or tissue under study. Embed tissue in OCT medium and snap-freeze by immersion into liquid nitrogen.Cut 4 mm frozen sections with a cryostat. Place sections on either albumin- or gelatin-coated slides.
Preparation of cells:
Pulse cells with 10 mM BrdU for 60 min. Cells grown on coverslips, or cytocentrifuge preparations made from cells grown in suspension, can be used for anti-bromodeoxyuridine staining according to the procedure below.
Procedure
1. Fix tissue sections or cells (on slide or coverglass) by immersing in absolute methanol for 10 minutes at +2-8°C. Air dry after removing from fixative. The slides can be stored at -20°C in a sealed box, or rehydrated to prepare for the assay procedure. To rehydrate, immerse in PBS for 3 min.
2. Denature DNA by incubating the slides in 2 N HCI for 60 min at +37°C.
3. Neutralize the acid by immersing the slides in 0.1 M borate buffer, pH 8.5. Change the buffer twice over a 10 min period.
4. Wash slides with PBS, changing the solution three times over a 10 min period.
5. Place slides in a humidified chamber (e.g., a sealed plastic box layered with wet paper towels) and cover cells with 150-300 μL of solution containing approximately 6 μg/mL anti-bromodeoxyuridine antibody diluted in PBS with 0.1% BSA. Incubate for 60 min at room temperature.
6. Wash slides with PBS, changing the solution three times over a 10 min period.
7. Apply optimal dilution of a second antibody conjugate (e.g., anti-mouse IgG-peroxidase), incubate, wash, and perform detection with a substrate that produces an insoluble product. After detection, counterstain with Harris-modified hematoxylin if desired. Slides can then be dehydrated and mounted.
Flow Cytometry: (0.2 μg/100 μl/10E6 cells) Optimal working dilutions must be determined by end user.
APPLICATIONS
Flow cytometry:The method below is based on that of M. Vanderlaan et al. (1986). Variations of this method exist in the literature, one consideration being the effect various fixation procedures have on the light-scattering properties of different cell populations. Procedure:
1. To label cells, pulse with 10 μM bromodeoxyuridine for 30 minutes. Harvest cells from culture.
2. Fix cells in 70% ethanol at +2-8°C for at least 30 min. Extract histones by resuspending cells in 1 mL chilled 0.1 M HCI containing 0.5% Triton X-100; incubate the suspension on ice for 10 minutes. Dilute acid with 5 mL distilled water and centrifuge at 200 x g for 10 min. Resuspend cells in 2 mL distilled water.
3. Denature cellular DNA by submerging the cell suspension into a boiling water bath for 10 min. Afterwards, quickly cool by placing the cell suspension in an ice slurry for several minutes. Wash cells in PBS that contains 0.5% Triton X-100.
4. Resuspend the cells (1-2 x 10 6 cells) in 100 μL of solution containing approximately 2 μg/mL anti-bromodeoxyuridine antibody diluted in PBS containing 0.1% BSA (0.2 μg/test). Incubate for 30 min at room temperature. Wash cells with PBS.
5. Resuspend cells in 100 μL of diluted goat anti-mouse IgG-FlTC Wash cells with PBS.
APPLICATIONS (Cont.)
Immunohistochemistry: Below is a procedure for staining cells that have been labeled with BrdU in vivo or in vitro. The procedure is based on the methods of B. Schutte et al. (1987) and D. Campana et al. (1988).
Preparation of tissue:
Inject animal with 50 mg BrdU/kg body weight. Sacrifice animal one hour later and remove organ or tissue under study. Embed tissue in OCT medium and snap-freeze by immersion into liquid nitrogen.Cut 4 mm frozen sections with a cryostat. Place sections on either albumin- or gelatin-coated slides.
Preparation of cells:
Pulse cells with 10 mM BrdU for 60 min. Cells grown on coverslips, or cytocentrifuge preparations made from cells grown in suspension, can be used for anti-bromodeoxyuridine staining according to the procedure below.
Procedure
1. Fix tissue sections or cells (on slide or coverglass) by immersing in absolute methanol for 10 minutes at +2-8°C. Air dry after removing from fixative. The slides can be stored at -20°C in a sealed box, or rehydrated to prepare for the assay procedure. To rehydrate, immerse in PBS for 3 min.
2. Denature DNA by incubating the slides in 2 N HCI for 60 min at +37°C.
3. Neutralize the acid by immersing the slides in 0.1 M borate buffer, pH 8.5. Change the buffer twice over a 10 min period.
4. Wash slides with PBS, changing the solution three times over a 10 min period.
5. Place slides in a humidified chamber (e.g., a sealed plastic box layered with wet paper towels) and cover cells with 150-300 μL of solution containing approximately 6 μg/mL anti-bromodeoxyuridine antibody diluted in PBS with 0.1% BSA. Incubate for 60 min at room temperature.
6. Wash slides with PBS, changing the solution three times over a 10 min period.
7. Apply optimal dilution of a second antibody conjugate (e.g., anti-mouse IgG-peroxidase), incubate, wash, and perform detection with a substrate that produces an insoluble product. After detection, counterstain with Harris-modified hematoxylin if desired. Slides can then be dehydrated and mounted.
Verwenden Sie Anti-BrdU-Antikörper, Klon AH4H7-1 / 131-14871 (monoklonaler Maus-Antikörper), der in FC, ICC, IHC validiert wurde, zum Nachweis von Bromdesoxyuridin, auch BrdU genannt.
Biochem/physiol Actions
Er bindet an Bromdesoxyuridin und kreuzreagiert mit Ioduridin (10 %). Anti-Bromdesoxyuridin kreuzreagiert weder mit Fluordesoxyuridin noch mit endogenen Zellbestandteilen wie Thymidin oder Uridin.
Physical form
Flüssigkeit in 10 mmol/l Phosphatpuffer, pH-Wert 7,4, mit 150 mmol/l NaCl und 0,1 % Natriumazid
Format: Aufgereinigt
Mit Protein A aufgereinigt
Preparation Note
Bei -20 °C ab Versanddatum 6 Monate haltbar. Aliquotieren, um wiederholtes Einfrieren und Auftauen zu vermeiden. Für maximale Produktrückgewinnung das Originalfläschchen nach dem Auftauen und vor dem Abnehmen der Kappe zentrifugieren.
Analysis Note
Kontrolle
Nach dem Einbringen von BrdU, alle DNA-enthaltenden Spezies
Nach dem Einbringen von BrdU, alle DNA-enthaltenden Spezies
Other Notes
Ersetzt: MAB1467
Konzentration: Die chargenspezifische Konzentration entnehmen Sie bitte dem Analysenzertifikat.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Sofern in unserem Katalog oder anderen Begleitdokumenten unserer Produkte nicht anders angegeben, sind unsere Produkte nur für Forschungszwecke vorgesehen und nicht für andere Zwecke zu verwenden, einschließlich, jedoch nicht beschränkt auf unautorisierte kommerzielle Verwendung, zur In-vitro-Diagnostik, für Ex-vivo- oder In-vivo-Therapiezwecke oder jegliche Art der Einnahme oder Anwendung bei Menschen oder Tieren.
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