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  • 產品名稱：WWEAffinityResinSet

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WWEAffinityResinSet
詳情介紹：
Specificity The WWE Affinity Resin, ABIN1741734 is highly purified GST-RNF146(100-175) fusion protein construct expressed in E. coli, and bound to glutathione beads.
WWE Negative Control Resin, ABIN1741735 is identical to the ABIN1741734 resin except for R163A substitution, which effectively abolishes PAR binding. The negative control resin is useful to control for non-specific binding, and its use is optional.
Both products will be delivered each in 0.5 mL (0.5 mg fusion protein) supplied as a slurry containing approx. 50 μL resin.
Characteristics Set contains one ABIN1741734 and ABIN1741735.
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, 0.1 % SDS, and protease inhibitors)
Cell/tissue extract containing approx. 0.15 to 1 mg total protein per sample
Microcentrifuge tubes
Microcentrifuge
SDS-PAGE sample buffer
Alternative Name RNF146
Sub Type Cocktail
Background RNF146 (Iduna) is a RING-domain E3 ubiquitin ligase that positively regulates Wnt signalling. RNF146 directly interacts with poly(ADP-ribose) through its WWE domain. The WWE domain is a conserved globular domain found in multiple PARPs and E3 ligases.
Molecular Weight 8 kDa + GST
Research Area Neurology, Alzheimer's Disease
Application Notes 20 μL=20 μg per reaction (for each)
Comment

280.00
Protocol 1. Resuspend the WWE 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. 0.5 mL of lysis buffer in a Microfuge tube.
3. Sediment resin at 10k x g in a Microfuge for 20 s. Carefully remove most of the lysis buffer, leaving the resin (barely visible) undisturbed in the tube. NOTE: Position the 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) 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 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 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 WWE Affinity Resin Set (ABIN1741736) Isolation of PARsylated PARP1 and TNKS1 using WWE resin. WWE and neg control resins w...
Product cited in: Zhong, Yeh, Hao, Pourtabatabaei, Mahata, Shao, Chessler, Chi: "Nutritional energy stimulates NAD+ production to promote tankyrase-mediated PARsylation in insulinoma cells." in: PLoS ONE, Vol. 10, Issue 4, pp. e0122948, 2015 (PubMed).

Background publications Zhou, Chan, Xiao, Tan: "Ring finger protein 146/Iduna is a poly(ADP-ribose) polymer binding and PARsylation dependent E3 ubiquitin ligase." in: Cell adhesion & migration, Vol. 5, Issue 6, pp. 463-71, 2012 (PubMed).

Wang, Deng, Zhang, Wang, Bian, Yin: "Systematic analysis of plant-specific B3 domain-containing proteins based on the genome resources of 11 sequenced species." in: Molecular biology reports, Vol. 39, Issue 5, pp. 6267-82, 2012 (PubMed).

Kang, Lee, Shin, Andrabi, Chi, Gagné, Lee, Ko, Lee, Poirier, Dawson, Dawson: "Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, Issue 34, pp. 14103-8, 2011 (PubMed).

Zhang, Liu, Mickanin, Feng, Charlat, Michaud, Schirle, Shi, Hild, Bauer, Myer, Finan, Porter, Huang, Cong: "RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling." in: Nature cell biology, Vol. 13, Issue 5, pp. 623-9, 2011 (PubMed).

Callow, Tran, Phu, Lau, Lee, Sandoval, Liu, Bheddah, Tao, Lill, Hongo, Davis, Kirkpatrick, Polakis, Costa: "Ubiquitin ligase RNF146 regulates tankyrase and Axin to promote Wnt signaling." in: PLoS ONE, Vol. 6, Issue 7, pp. e22595, 2011 (PubMed).

Andrabi, Kang, Haince, Lee, Zhang, Chi, West, Koehler, Poirier, Dawson, Dawson: "Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death." in: Nature medicine, Vol. 17, Issue 6, pp. 692-9, 2011 (PubMed).