Protein Kinase, DNA-Activated, Catalytic Polypeptide (PRKDC) (pT 2609) antibody

Antigen: Protein Kinase, DNA-Activated, Catalytic Polypeptide (PRKDC)

Synonyms: HYRC, p350, DNAPK, DNPK1, HYRC1, XRCC7, DNA-PKcs, prkdc-A, dna-pkcs, PRKDC, DNA-PKCS

Epitope: pT 2609

Clonality: Polyclonal

Host: Rabbit

Reactivity: Human, Chimpanzee

Application: ELISA, Western Blotting (WB)

Catalog no.: ABIN129603

Additional Information

Alternative name: DNA Dependent Protein Kinase (DNA PKcs pT 2609)

Gene ID: NP_008835

Swiss-Prot: P78527

Immunogen: This affinity purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to amino acids surrounding Thr 2609 of human DNA PKcs.

Format: Liquid (sterile filtered)

Description: DNA dependent Protein Kinase (also called DNAPK, DNPK1, HYRC1, Protein Kinase DNA Activated Catalytic Polypeptide, XRCC7 and p460) consists of the 460 kDa DNA PKcs and a heterodimeric regulatory complex comprised of p70 Ku and p80 Ku (Ku autoantigen). DNA PKcs is a nuclear protein serine/threonine kinase present in a wide variety of eukaryotic species. DNA PKcs phosphorylates transcription factors, Sp1, Oct-1, p53 and SV40 large T antigen. DNA PKcs is involved in repairing double stranded DNA breaks. At the onset of apoptosis, DNA PKcs is rapidly inactivated by cleavage of the catalytic subunit into smaller polypeptides. Proteolysis of DNA PKcs is inhibited by the cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. Alternative splicing can occur for this protein to produce at least two isoforms.

Specificity: This affinity purified antibody is directed against the phosphorylated form of human DNA PKcs at the pT2609 residue. The product was affinity purified from monospecific antiserum by immunoaffinity purification. Antiserum was first purified against the phosphorylated form of the immunizing peptide. The resultant affinity purified antibody was then cross-adsorbed against the non-phosphorylated form of the immunizing peptide. This phospho-specific polyclonal antibody reacts with phosphorylated pT2609 of human DNA PKcs. Reactivity with non-phosphorylated human DNA PKcs is minimal. A BLAST analysis was used to suggest reactivity with this protein from human and chimpanzee based on 100% homology for the immunogen sequence. However, cross-reactivity is expected with mouse, rat, dog, chicken and horse DNA PKcs based on a high degree of homology to the immunogen sequence. Cross-reactivity with DNA PKcs homologues from other sources has not been determined.

Application Details

Application Notes: This affinity purified antibody has been tested for use in ELISA and by western blot. Specific conditions for reactivity should be optimized by the end user. Expect a band approximately 460 kDa in size corresponding to DNA PKcs by western blotting in the appropriate cell lysate or extract. Alternate splice variants have been described for this protein. Best western blotting results are seen when IP is performed prior to detection. This antibody detects an inducible signal at the correct height after DNA damage. However, the antibody may also detect an IR-inducible signal in cells lacking DNA PKcs (MO59J cells) at the same size as DNA PKcs if western blotting is performed directly. We believe that this additional band is 53BP1 that runs at the same size as DNA PKcs and is also phosphorylated in an IR-dependent manner. Similar results were seen with another phospho DNA PKcs antibody indicating that this result is general to antibodies to this phospho site, rather than specific to this antibody. ELISA: 1:300,000. WB: 1:500 - 1:2,000. OTHER: User Optimized.

Concentration: 1.1 mg/ml (by UV absorbance at 280 nm)

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 0.01% (w/v) Sodium Azide

Storage: Store vial at -20° C prior to opening. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use. For extended storage aliquot contents and freeze at -20° C or below. Avoid cycles of freezing and thawing. Expiration date is one (1) year from date of opening product.

Restrictions: For Research Use only

Images

Western blot using antibodies-online's Affinity Purified anti-DNAPKcs antibody shows detection of a 460 kDa band corresponding to human DNAPKcs in various preparations. Lane 1: Fus1 untreated, Lane 2: Fus1 IR (20Gy, 4h), Lane 3: Fus1 DNAPK inhibitor + IR, Lane 4: MO59J (DNAPK-) untreated, Lane 5: MO59J IR, Lane 6: Fus1 untreated, Lane 7: Fus1 IR (20Gy, 4h), Lane 8: Fus1 DNAPK inhibitor + IR, Lane 9: MO59J untreated, Lane 10: MO59J IR. Lanes 1-5 are nuclear extract whereas lanes 6-10 are whole cell lysates. MO59J is a cell line that lacks DNAPKcs. FUS1 is the matched cell line complemented with a chromosomal fragment containing the DNA-PKcs gene. Approximately 20 ug of lysate was run on SDS-PAGE and transferred onto nitrocellulose, followed by reaction with a 1:1,000 dilution of anti-DNAPKcs antibody. Detection occurred using a 1:5,000 dilution of HRP-labeled Goat anti-Rabbit IgG for 1 hour at room temperature. A chemiluminescence system was used for signal detection (Roche) using a 1 min exposure time.

Publications

Publications: Dittmann, Mayer, Wanner et al.: "The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation." in: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, Vol. 84, Issue 3, pp. 328-34, 2007 (PubMed).

Dittmann, Mayer, Ohneseit et al.: "Celecoxib induced tumor cell radiosensitization by inhibiting radiation induced nuclear EGFR transport and DNA-repair: a COX-2 independent mechanism." in: International journal of radiation oncology, biology, physics, Vol. 70, Issue 1, pp. 203-12, 2007 (PubMed).

Wanner, Mayer, Kehlbach et al.: "Activation of protein kinase Cepsilon stimulates DNA-repair via epidermal growth factor receptor nuclear accumulation." in: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, Vol. 86, Issue 3, pp. 383-90, 2008 (PubMed).

Dittmann, Mayer, Kehlbach et al.: "The radioprotector Bowman-Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorylation and nuclear transport." in: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, Vol. 86, Issue 3, pp. 375-82, 2008 (PubMed).

Yoshikawa, Kashino, Ono et al.: "Phosphorylated H2AX foci in tumor cells have no correlation with their radiation sensitivities." in: Journal of radiation research, Vol. 50, Issue 2, pp. 151-60, 2009 (PubMed).