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Active Motif's DNA Repair Protein ELISAs are DNA-binding ELISAs that detect activity of DNA repair proteins in both mammalian tissue and cell culture extracts. The kits combine a fast, user-friendly format that is more sensitive than gelshift while eliminating the use of radioactivity and providing quantitative results in less than five hours. For more information on how they work, click on the Method tab below.
The RPA DNA Repair Protein ELISA was designed to study regulation of the Replication Protein A, which is composed of RPA70, RPA32 and RPA14. It contains a 96-well plate to which a single-stranded linear DNA molecule has been immobilized. RPA in nuclear extracts binds specifically to this DNA molecule where it is detected and quantitated by addition of either the included RPA32 antibody, followed by HRP-conjugated secondary antibody and developing solution. Click the RPA Info tab below for data and more information.
| Name | Format | Cat No. | Price | |
|---|---|---|---|---|
| RPA DNA Repair Protein Kit | 1 x 96 rxns | 51296 | $665 | Buy Now |
| 5 x 96 rxns | 51796 | $2,795 | Buy Now |
| DNA Repair RPA Manual |
| DNA Repair Profile |
| Cell Biology Products Brochure |
| MSDS: Sodium Azide |
| MSDS: Sulphuric Acid |
| MSDS: Thimersol |
Replication Protein A (RPA) is composed of RPA70, RPA32 and RPA14 (70, 32 and 14 kDa subunits, respectively). RPA binds directly to single-stranded DNA (ssDNA) where it organizes and protects ssDNA during DNA replication, recombination and repair. The inability to replicate or repair DNA breaks leads to cell cycle arrest and apoptosis.
Applications
RPA is involved in the maintenance of genomic stability and therefore represents an excellent pharmacological target for developing drugs to treat cancer. Applications of the RPA DNA Repair Kit include:
- Profiling of RPA32 under various growth and stimulation conditions
- Drug screening and/or potency toward RPA
- RPA activity regulation
- Protein structure/function studies of RPA
Figure 1: Monitoring RPA32 binding with the RPA DNA Repair Kit.
Different amounts of nuclear extracts from unstimulated Raji and Jurkat cells are tested for activity using the Ku70/86 DNA Repair Kit and its RPA32 antibody. These curves are provided for demonstration only.
DNA repair proteins act to maintain genome integrity by recognizing, binding to and repairing damaged DNA. Deficiencies in the activity of these proteins are linked to the development of many pathological diseases, including cancer. Understanding and quantifying DNA repair proteins can help elucidate the molecular mechanisms of DNA damage and repair pathways, and understand the damage specificity of a repair protein.
The DNA Repair Protein ELISA Kit advantage
DNA Repair Protein ELISAs offer an improved method for studying DNA repair protein activity. Current techniques used to study DNA damage and repair include EMSAs and Western blots, which are time consuming, do not allow for high-throughput and provide only semi-quantitative results. In contrast, Active Motif’s DNA Repair Kits are DNA-binding ELISAs that eliminate radioactivity and provide quantitative results in less than five hours.
Each kit includes a 96-well plate in which multiple copies of a specific damaged oligonucleotide have been immobilized. When cellular extract is added, the repair protein of interest binds to the damaged DNA. Each well is then incubated with a primary antibody that is specific for the repair protein being studied. Addition of a secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout (Figure 1). Also included are wild-type (DNA lesion cotaining) and mutated oligonucleotides that can be used in competiton assays to verify the specificity of the signal (optional).
Key features
- Non-radioactive, colorimetric method
- Quantitative results in less than 5 hours
- 10X more sensitive than gelshift
- High and low-throughput compatible – 96-stripwell format
- Ability to assay both cells and tissues
Figure 1: Flow chart of DNA Repair Protein ELISAs.
DNA repair proteins bind to oligonucleotides that are immobilized in the well. Incubation with primary & secondary antibodies and developing solution enables accurate measurement of DNA repair protein activity using colorimetric readout.
Contents & Storage
One or five 96-well assay plates with plate sealers, RPA32 primary antibody, HRP-conjugated secondary antibody, competitor oligonucleotide, positive control cell extract, DTT, Protease Inhibitor Cocktail, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffer, and Developing and Stop Solutions. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.
