Poly-ADP-ribose Affinity Resin Set

Details for Product No. ABIN1741733, Supplier: Log in to see
Target Name (Antigen)
Reactivity
Archaeoglobus fulgidus
Host
Escherichia coli (E. coli)
Antibody Type
Recombinant
Conjugate
GST tag
Application
Pull-Down Assay (Pull-Down)
Options
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Specificity The PAR Affinity Resin, ABIN1741731 is highly purified GST-Af1521 macrodomain fusion protein construct expressed in E. coli, and bound to glutathione beads. The Af1521 macrodomain protein is reported to also bind mono-ADP-ribosylated proteins and ADP-ribose.
PAR Negative Control Resin, Cat. ABIN1741732 is identical to the ABIN1741731 resin except for two gly to asp substitutions, which abolish PAR binding. The negative control resin is useful to control for non-specific binding, and its use is optional.
Both products are supplied as 1 mL slurry containing approx. 75 µL resin (1 mg fusion protein)
Characteristics Set contains one ABIN1741731 and ABIN1741732.
Purification Affinity chromatography
Purity > 95 %
Material not included Lysis buffer (e.g.: 50 mM Tris, pH 8, 200 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 10 % glycerol, 1 mM DTT, 0.5 % deoxycholate, and protease inhibitors)
Cell/tissue extract containing approx. 0.15 to 1 mg total protein per sample
Microcentrifuge tubes
Microcentrifuge
SDS-PAGE sample buffer
Sub Type Cocktail
Background Af1521 is a thermopohilic protein from Archaeoglobus fulgidus, and contains a conserved approx. 190 AA domain known as the macro domain. Macrodomains are found in a wide variety of organisms including bacteria, viruses, and vertebrates. Expressed and purified macrodomains from Af1521, Alc1, macroH2A and Bal/PARP9 proteins have been shown to bind polymeric ADP-ribose modified proteins with high specificity and affinity
Molecular Weight 21 kDa + GST
Application Notes 20 µL=20 µg per reaction (for each)
Comment

280.00

Protocol 1. Resuspend the PAR affinity and neg control resins by gently inverting the product tubes several times to obtain a homogenous suspension of resin.
2. Use a wide-bore pipette or a cut pipette tip to transfer 20 µL of the suspension to approx. 1 mL of lysis buffer in a Microfuge tube.
3. Sediment resin at 15k x g in a Microfuge (highest speed setting) for 20 s. Carefully remove most of the lysis buffer, leaving the resin (barely visible) undisturbed in the tube.
NOTE: Position tubes in the Microfuge with the hinge oriented outward in order to ascertain the location of the sedimented resin.
4. Add cell/tissue extract in lysis buffer to the Microfuge tube containing the resin. Suggested extract protein amount is 0.15 to 1 mg in a total buffer volume of 0.5 mL.
5. Incubate the reaction for several hours or overnight at 4 °C on a Nutator or similar device.
6. Sediment then wash resin 3-times with 0.5-1 mL lysis buffer, as in step 3. On the final wash, carefully remove residual buffer without disturbing the resin.
7. Add 75 µL 1X SDS-PAGE sample buffer to each tube, agitate, then incubate at 95 °C for 10 min to dissociate GST-macrodomain from PARylated proteins and the resin.
8. Run samples on SDS-PAGE, and perform Western blotting. Probe immunoblot using desired protein-specific antibodies, for example anti-PARP1 (ABIN1741708), or anti-poly-ADP-ribose antibodies (ABIN1741702 or ABIN1741707) to detect affinity purified proteins. Compare results to negative control resin samples to assess non-specific binding, which should be minimal.
Restrictions For Research Use only
Format Liquid
Buffer PBS with 1 mM EDTA, 1 % Triton X-100, and 0.02 % sodium azide.
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice Do not freeze!
Storage 4 °C
Storage Comment Stable for 6 months from date of shipment when stored at 4 ˚C.
Supplier Images
Western Blotting (WB) image for Poly-ADP-ribose Affinity Resin Set (ABIN1741733) Western blot of Poly-ADP-ribosylated PARP1 and TNKS1 in MDCK cells using PAR-affinity...
Western Blotting (WB) image for Poly-ADP-ribose Affinity Resin Set (ABIN1741733) Pull-down of Poly-ADP-ribosylated PARP1 by PAR-affinity resin. Purified poly-ADP-ribo...
Product cited in: Gagné, Pic, Isabelle, Krietsch, Ethier, Paquet, Kelly, Boutin, Moon, Foster, Poirier: "Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress." in: Nucleic acids research, Vol. 40, Issue 16, pp. 7788-805, 2012 (PubMed).

Background publications Timinszky, Till, Hassa, Hothorn, Kustatscher, Nijmeijer, Colombelli, Altmeyer, Stelzer, Scheffzek, Hottiger, Ladurner: "A macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation." in: Nature structural & molecular biology, Vol. 16, Issue 9, pp. 923-9, 2009 (PubMed).

Gottschalk, Timinszky, Kong, Jin, Cai, Swanson, Washburn, Florens, Ladurner, Conaway, Conaway: "Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, Issue 33, pp. 13770-4, 2009 (PubMed).

Dani, Stilla, Marchegiani, Tamburro, Till, Ladurner, Corda, Di Girolamo: "Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, Issue 11, pp. 4243-8, 2009 (PubMed).

Karras, Kustatscher, Buhecha, Allen, Pugieux, Sait, Bycroft, Ladurner: "The macro domain is an ADP-ribose binding module." in: The EMBO journal, Vol. 24, Issue 11, pp. 1911-20, 2005 (PubMed).