M30 CytoDEATH

Кат. номер

12140349001

12140322001

Mouse monoclonal antibody (Clone M30, mouse IgG2b) is used for the detection of apoptosis in epithelial cells (caspase cleavage product of cytokeratin 18). It is provided in lyophilized, stabilized, formalin grade form.
During apoptosis, vital intracellular proteins are cleaved. The proteases that mediate this process are called caspases (Cysteinyl-aspartic acid proteases). Caspases are expressed as zymogenes, which are activated by different apoptosis inducers. Once activated, a single caspase activates a cascade of caspases. Until recently, cytokeratins, in particular cytokeratin 18 (CK18), were not known to be affected by early events of apoptosis. Recently, it has been shown that the M30 antibody recognizes a specific caspase-cleavage site within cytokeratin 18, which is not detectable in native CK18 of normal cells. Consequently, the M30 CytoDEATH antibody is a unique tool for the easy and reliable determination of very early apoptotic events in single cells and tissue sections.

• Assay time: 2 hours for immunofluorescence on cells, 3.5 hours for staining of tissues (excluding dewaxing)
• Sample material: Adherent cells, tissue samples (routinely fixed and paraffin-embedded tissue sections, cryostat sections)

Specificity
The M30 CytoDEATH antibody binds to a caspase-cleaved, formalin-resistant epitope of the cytokeratin 18 (CK 18) cytoskeletal protein. The immunoreactivity of the antibody is confined to the cytoplasm of apoptotic cells.
The M30 CytoDEATH antibody is used for the determination of early apoptotic events in cells and tissue sections by detection of a specific epitope of cytokeratin 18 that is presented after cleavage by caspases. Detect apoptosis by applying the M30 antibody to fixed samples, then using secondary detection systems suitable for immunohistochemistry, immunocytochemistry, and flow cytometry.
Features and Benefits

• Convenient: Easy-to-use standard protocol
• Clear results: Apoptotic cells are most easily distinguishable from normal cells
• Early detection of apoptosis: Detects caspase-cleaved Cytokeratin 18; caspase activity is one of the earliest apoptosis markers
• Useful for paraffin-embedded tissue: Useful for routinely fixed tissue samples; retrograde studies are possible

Physical form
White lyophilizate. The antibody has been lyophilized in the presence of proteinous stabilizers.
Preparation Note
Short protocol
For formalin-embedded tissue:

• Dewax formalin-fixed, paraffin-embedded tissue sections.
• Retrieve antigen by heating in citric-acid buffer.
• Add M30 antibody.
• Add Anti-mouse-biotin.
• Add Streptavidin-POD.
• Add substrate solution (DAB or AEC).
• Counterstain with Harris hematoxylin.
• Analyze under a light microscope.

For immunofluorescence on cells:

• Fix cells.
• Add M30 antibody.
• Add Anti-mouse-Ig-fluorescein.
• Analyze under a fluorescence microscope.

Working concentration: The concentration of the stock solution is 6,6 ng/ µl. For the QC-release test of this product, a function test using a cellular model (HeLa cells treated with TNF-/Actinomycin D) is performed, as mentioned in the pack insert. The quantity of protein is only part of the QC-release test of the stock product that is why it is not included in the documentation.
Working solution: Procedure for Immunofluorescence in Cells and Flow Cytometry
When using other detection methods or sample material, conditions may vary and have to be adapted.
For application, dilute the antibody stock solution in Incubation buffer (M30 CytoDEATH).
Note: The antibody solutions should be free of precipitate. If necessary, centrifuge the solutions at high speed prior to use.
Additional working solutions required for the immunofluorescence and flow cytometry procedure:
Washing buffer: PBS containing 0.1% Tween 20
Incubation buffer: PBS containing BSA and 0.1% Tween 20
Procedure for Immunohistochemistry

When using other detection methods or sample material, the conditions may vary and have to be adapted.
Dilute the antibody stock solution in Incubation buffer.
Note: The antibody solutions should be free of precipitate. If necessary, centrifuge the solution at high speed prior to use.
Working solutions need to perform the immunohistochemistry staining procedure:
Washing buffer: PBS containing 0.1% Tween 20
Incubation buffer: PBS containing BSA and 0.1% Tween 20
Citric acid buffer: 2 g/l citric acid, pH 6 adjusted with 1 N NaOH
Storage conditions (working solution): The antibody stock solution is stable for 6 months at 2 to 8 °C. Alternatively, it can be stored in aliquots at -15 to -25 °C.
Note: Avoid repeated freezing and thawing.
Reconstitution
Reconstitute lyophilizate in 550 μl to obtain an antibody stock solution.
For life science research only. Not for use in diagnostic procedures.
The M30 antibody is made under a license agreement from Peviva AB, Sweden.

Quality Level	100,
form	lyophilized (clear, colorless solution after reconstitution)
usage	sufficient for 250 tests (12140349001), sufficient for 50 tests (12140322001)
Торговая марка	Roche
shipped in	wet ice
storage temp.	2-8°C

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NONH for all modes of transport