ssDNA Apoptosis ELISA Kit

This procedure is based on the selective denaturation of DNA in apoptotic cells by formamide, and detection of denatured DNA with monoclonal antibody to single-stranded DNA (ssDNA). Formamide is a gentle agent that denatures DNA in apoptotic cells, but not in necrotic cells or in the cells with DNA breaks in the absence of apoptosis (Frankfurt & Krishan 2001). The sensitivity of DNA in apoptotic cells to formamide is not related to DNA breaks, but rather reflects changes in chromatin associated with apoptosis, such as chromatin condensation and digestion of proteins stabilizing DNA. The assay includes attachment of cells to 96-well plates, treatment of attached cells with formamide, and staining of ssDNA in apoptotic cells with a mixture of primary antibody and peroxidase-labeled secondary antibody. The protocol based on the one-step detection of ssDNA with antibody mixture has higher sensitivity and lower number of steps than standard two-step immunostaining (Frankfurt & Krishan 2001). This mixture is included in the kit in a ready to use form.

Application:

The CHEMICON ssDNA Apoptosis ELISA Kit is a convenient, sensitive method for early detection of apoptosis. The assay can be performed in two formats:

1. Cells are grown, treated with apoptosis inducing agents, stained and analyzed in the same microtiter plate. This assay is suitable for high throughput screening when induction of apoptosis is used as the endpoint of drug activity.

2. Cell suspensions obtained from cultures or tissues are transferred into a microtiter plate for staining and analysis. For this assay, cells could be fixed with methanol and stored before transfer into plates. Alternatively, fixation could be performed in microtiter plates after transfer of non-fixed cells.

Total assay time is 3-4 hours, which includes fixation and staining. Only one washing step is required.

The ssDNA Apoptosis ELISA is for research use only. Not for use in diagnostic or therapeutic procedures.

Positive Control

To use the ssDNA positive control included in this kit:

1. Add 100 μL of ssDNA Positive Control solution per well and dry plate by floating in a 37ºC waterbath or in a 37ºC incubator or culture hood (fan on) with lid off, overnight or until very dry. WELLS MUST BE THROUGHLY DRY for ssDNA to adhere to the wells.

2. Rinse wells with PBS before use. Plate can be stored dry, covered until ready to use.

3. Proceed to step 8 of Assay Instructions below if positive control is done on a separate plate. If performing entire assay on one plate, proceed to sample preparation below, ignoring wells used for positive control UNTIL step 8.

Note: Absorbance reading between 1.5 and 2.8 indicates good assay sensitivity; lower values will still work, however it may indicate that some of the ssDNA has come off the plate. One can reapply ssDNA standard again, if necessary.

Research Category
Apoptosis & Cancer

Used to detect/quantify: ssDNA

96 wells

Store kit materials at -20°C. Once the kit is opened, Antibody Mixture and ssDNA Positive Control should be thawed on ice, aliquoted and stored at -20ºC. Formamide, Wash Buffer, ABTS Solution and Stop Solution can be thawed and stored at 4°C.

Antibody Mixture, Primary monoclonal to ssDNA and HRP-labeled goat anti-mouse IgM (Part No. 71278a): One 10 mL bottle, premixed (Ready to Use).

Formamide (Part No. 71278b): One 5 mL bottle (Ready to Use).

Single-stranded DNA Positive Control (Part No. 71278c): One 2 mL vial at 0.3 μg/mL.

Wash Buffer Concentrate (Part No. 90160): One 10 mL (10X) bottle of Concentrate.

ABTS Solution (Part No. 90161): One 12 mL bottle of a Ready to Use solution of 2,2′-AZINO-bis [3-ethylbenziazoline-6-sulfonic acid] in a proprietary buffer with enhancer.

Stop Solution (Part No. 90162): One 12 mL bottle of an HCl solution (Ready to Use).

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.