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512729 PARP Cleavage Detection Kit

PARP Cleavage Detection Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
512729
  
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      Overview

      Replacement Information
      Description
      Overview

      This product has been discontinued.





      This product has been discontinued.



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      An assay kit designed to detect poly(ADP-ribose) polymerase (PARP) cleavage by immunoblotting. PARP is an enzyme implicated in DNA damage and repair mechanisms. During apoptosis, PARP is cleaved by caspase-3. Cleavage of PARP from the native 116 kDa to 85 kDa is a hallmark of apoptosis. Each kit is provided with a highly specific rabbit polyclonal antibody that detects 116 kDa PARP and the 85 kDa apoptosis-related cleavage fragment from human, bovine, rat, and mouse.
      Catalogue Number512729
      Brand Family Calbiochem®
      References
      ReferencesDuriez, P.J., et al. 1997. Biochim. Biophys. Acta 1334, 65.
      Lazebnik, Y.A., et al. 1994. Nature 371, 346.
      Kaufman, S., et al. 1993. Cancer Research. 53, 3976.
      Lamarre, D., et al. 1988. Biochim. Biophys. Acta 950, 147.
      Product Information
      Detection methodImmunoblot
      FormatImmunoblot
      Kit containsA Control Blocking Peptide, Uninduced Immunoblotting Standard, Induced Immunoblotting Standard, PARP Immunoblotting Standard, and a user protocol.
      Applications
      Biological Information
      Assay time5.5-6 h
      Primary Targetdetect poly(ADP-ribose) polymerase (PARP) cleavage by immunoblotting
      Sample TypeCell extracts
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20/22-24-36/37/38-40-41

      Harmful by inhalation and if swallowed.
      Toxic in contact with skin.
      Irritating to eyes, respiratory system and skin.
      Limited evidence of a carcinogenic effect.
      Risk of serious damage to eyes.
      S PhraseS: 26-36/37/39-45-53

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Avoid exposure - obtain special instructions before use.
      Product Usage Statements
      Intended useUpon arrival store the entire contents of the kit at -70°C for the highest stability.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsA Control Blocking Peptide, Uninduced Immunoblotting Standard, Induced Immunoblotting Standard, PARP Immunoblotting Standard, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      512729 0

      Documentation

      PARP Cleavage Detection Kit Certificates of Analysis

      TitleLot Number
      512729

      References

      Reference overview
      Duriez, P.J., et al. 1997. Biochim. Biophys. Acta 1334, 65.
      Lazebnik, Y.A., et al. 1994. Nature 371, 346.
      Kaufman, S., et al. 1993. Cancer Research. 53, 3976.
      Lamarre, D., et al. 1988. Biochim. Biophys. Acta 950, 147.

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision27-July-2010 RFH
      FormatImmunoblot
      Detection methodImmunoblot
      Speciesbovine, human, mouse, rat
      Intended useUpon arrival store the entire contents of the kit at -70°C for the highest stability.
      BackgroundPoly(ADP-ribose) polymerase (PARP) is an enzyme involved in DNA damage and repair mechanisms. It synthesizes poly(ADP-ribose) polymer onto chromatin and other target proteins. During apoptosis, PARP is cleaved by the protease caspase-3, an important downstream apoptotic caspase. Cleavage of PARP from the native 116 kDa to 85 kDa is a hallmark of apoptosis.
      Principles of the assayThe PARP Cleavage Detection Kit is designed to detect PARP cleavage by the method of immunoblotting. The kit includes a highly specific rabbit polyclonal antibody which detects the 116 kDa PARP and the 85 kDa apoptosis-related cleavage fragment from human, bovine, rat, and mouse. The kit also contains induced and non-induced cell extracts, PARP protein standard, and control blocking peptide.
      Materials provided• Anti-Poly(ADP-Ribose) Polymerase (PARP), (Rabbit), (Kit Component No. KP8501): Supplied as 25 µl rabbit serum containing 0.05% sodium azide in a screw-cap microcentrifuge tube. This antibody is stable at 4°C for short term storage. For long term storage, aliquot product into individual tubes and freeze at -20°C or -70°C. AVOID FREEZE/THAW CYCLES.
      • Control Blocking Peptide, (Kit Component No. KP8502): Supplied as 100 µg/vial, lyophilized solid, in a screw-cap microcentrifuge tube. Purity: ≥90% by HPLC. M.W. 1963.3. Store desiccated at -20°C or -70°C. After reconstitution with 100 µl dH₂O, store at -70°C.
      • Immunoblotting Standard, Uninduced, (Kit Component No. KP8503): Supplied as 200 µl of a whole cell extract of human HL60 leukemia cells, in a screw-cap microcentrifuge tube in SDS-PAGE sample buffer (62.5 mM Tris/HCl, pH 6.8; 6 M urea; 10% glycerol; 2% SDS; 0.00125% bromophenol blue; 5% β-mercaptoethanol). Uncleaved PARP, M.W: 116kDa. Store at -20°C or -70°C.
      USAGE: Ready for use on SDS-PAGE. Use ~20 µl sample (~75,000 cells) per lane for SDS-PAGE.
      • Immunoblotting Standard, Induced, (Kit Component No. KP8504): Supplied in SDS-PAGE sample buffer (see KP8503) as 200 µl of whole cell extract of human HL60 leukemia cells, induced to undergo apoptosis, by the chemotherapeutic agent etoposide. PARP uncleaved: M.W. 116 kDa; cleaved: M.W. 85 kDa as detected by KP8501. Store at -20°C or -70°C.
      USAGE: Ready for use on SDS-PAGE. Use approximately 20 µl sample (~75,000 cells) per lane for SDS-PAGE.
      • PARP (Poly (ADP-Ribose) Polymerase) Immunoblotting Standard, BovineThymus, (Kit Component No. KP8505): Supplied as 100 µl of PARP enzyme, ~10 µg/ml, in SDS-PAGE sample buffer. Store at -20°C or -70°C.
      USAGE: Ready for use on SDS-PAGE. Use ~10 µl sample per lane for SDS-PAGE. A band with M.W. 116 kDa, which represents uncleaved PARP, is detected by immunoblotting.
      Detailed protocolA. ImmunoBlotting using Anti-PARP Antibody, HL60 Cell Extracts, and PARP Immunoblotting Standard.

      1. HL60 cell extracts were prepared from exponentially growing cells either induced (for apoptosis), with or without etoposide (induced standard or uninduced standard respectively). Cells were washed once with PBS, suspended at ~4X106 cells/ml in sample buffer (6 M urea, 62.5 mM Tris/HCl; 10% glycerol, 5% b-mercaptoethanol, 2% SDS; 0.00125% bromophenol blue; pH 6.8), sonicated for 15 sec, and incubated at 65°C for 15 min.
      NOTE: The purpose of the urea in the sample buffer and the sonication step in the above protocol is to effectively dissociate PARP/DNA interactions. For preparing PARP electrophoresis samples from tissue see : Shah, G.M., et al. 1995. Anal. Biochem. 227, 1; Simonin, F., et al. 1991. Anal. Biochem. 195, 226.
      2. Run SDS-PAGE using 20 µl of control and induced HL60 cell extract and ~10 µl of PARP immunoblotting standard.
      3. Transfer proteins to nitrocellulose blotting membrane.
      4. Block nitrocellulose with "Blotto" [5% non-fat dry milk in TBST (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% TWEEN®-20 Detergent)] for 1 h at RT with gentle agitation.
      5. Incubate nitrocellulose with 1/500 dilution Anti-PARP in Blotto for 2.5 h at RT.
      6. Wash membrane with TBST X 3, 10 min per wash.
      7. Incubate nitrocellulose with secondary antibody (1/2000 in Blotto) using Anti-Rabbit Alkaline Phosphatase conjugate for 1 h at RT.
      8. Wash membrane with TBST 30 min with 3 changes of buffer. Rinse briefly with TBS (TBST without TWEEN®-20 detergent).
      9. Incubate with BCIP/NBT color development reagent until bands are desired darkness (~5 min with BCIP/NBT plus from Moss, Inc.). Rinse with TBS plus 20 mM EDTA to stop color development. NOTE: color development times may vary.

      NOTE: These procedures are intended only as a guide. The optimal concentrations of primary antibody and the experimental conditions must be determined by the individual user. No warranty or guarantee of performance using these procedures is made or implied.

      B. Antibody Pre-adsorption Using Control Peptide for Anti-PARP Antibody to Block Specific Staining of Anti-PARP.

      1. Reconstitute control (blocking) peptide with 100 µl dH2O to obtain a 1 mg/ml solution. Vortex thoroughly and allow >10 min to dissolve completely.
      2. In a plastic microcentrifuge tube, add the following (scale as necessary):
      a) 2 µl antibody
      b) 8 µl of control peptide (1 mg/ml)
      c) Bring total volume up to 30 µl with Tris-buffered saline
      3. Incubate at 4°C for 1 h with gentle agitation.
      4. Spin sample in microfuge at maximum speed for 15 min to pellet aggregates. Remove supernatant carefully to avoid disturbing any pellet.
      5. Make final dilutions with the supernatant as appropriate and use immediately for immunochemical method.

      NOTE: The above protocol was tested for immunoblotting. It is intended only as a guide. The optimal blocking conditions, particularly for other applications, may differ and must be determined by each user. Increasing the peptide/antibody ratio and the length of the binding incubation (Step 3) are two variations that may improve blocking.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Tween® is a registered trademark of ICI Americas, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.