WWE Protein

WWE Affinity Resin Set

Cat. Num. AAA376021

• Applications: Western Blot, Immunoblot, Western Blot
• Purity: GST-WWE fusion protein, purity >95% by SDSPAGE
• Synonyms: WWE; WWE Affinity Resin Set; WWE protein

• Purity/Purification: GST-WWE fusion protein, purity >95% by SDSPAGE
• Form/Format: Each vial contains 0.5mg purified GST-WWE fusion protein bound to 50-75ìL packed volume of glutathione beads in 0.5 mL buffer containing 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.02% sodium azide.

• Applicable Applications for WWE protein: Western Blot (WB), Immunoblot (IB)
• Application Notes: Use 20uL (20ug) suspended resin to affinity purify/pull-down poly-ADP-ribose modified proteins in 0.15-1mg cell and tissue extracts. Analyze by Western blotting using protein-specific antibodies to probe the immunoblot. Each 0.5mL vial is sufficient for analysis of ~25 samples.; Optimal experimental conditions must be determined by the user.
• Additional Notes: The WWE Neg Control Resin (MBS376023) has been shown to bind to purified highly automodified PARP1 under certain conditions.
• Materials Required: Lysis buffer (e.g.: 50mM Tris, pH 8, 200mM NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM DTT, 0.1% SDS, and protease inhibitors); Cell/tissue extract containing ~0.15 to 1mg total protein per sample; Microcentrifuge tubes; Microcentrifuge; SDS-PAGE sample buffer
• Procedure: 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 20µL of the suspension to ~0.5mL of lysis buffer in a microfuge tube.; 3. Sediment resin at 10k x g in a microfuge for 20 sec. 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 1mg in a total buffer volume of 0.5mL.; 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 0.5-1mL lysis buffer, as in step 3. On the final wash, carefully remove residual buffer without disturbing the resin.; 7. Add 75µL 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 (MBS376008) to detect affinity purified proteins. Compare results to negative control resin samples to assess non-specific binding, which should be minimal.
• Procedure: 1. HEK293 cells were grown to confluence on 10 cm plates.; 2. Cells were harvested at 4°C in 1 mL Lysis buffer (50mM Tris, pH 8, 200mM NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM DTT, 0.1% SDS, and protease inhibitors).; 3. After clarification by centrifugation at 15k x g for 10 min, cell lysates (~0.25mL) were incubated with WWE affinity resin (MBS376022) or neg control resin (MBS376023) (20µL suspended resin; see Suggested General Protocol) with agitation at 4°C overnight.; 4. Resins were sedimented with associated proteins in a microfuge at 15k x g for 10 sec, and supernatant discarded.; 5. Resin was washed 4-times with 500µL Lysis buffer.; 6. 100µL SDS-PAGE sample buffer was added to each tube then samples boiled for 10 min.; 7. SDS-PAGE and Western blotting of samples were performed. Immunoblots were probed with anti-PARP1 and anti-TNKS antibodies, and detected using ECL.
• Preparation and Storage: Stable for 6 months from date of shipment when stored at 4°C. DO NOT FREEZE!

Testing Data

(Isolation of PARsylated PARP1 and TNKS1 using WWE resin. WWE and neg control resins were used to pull down PARsylated proteins from clarified extracts of confluent HEK293 cells. The resin bound proteins were Western blotted and probed with either anti-TNKS1 or PARP1)

Related Product Information for WWE protein

Background: WWE Affinity Resin and Negative Control Resin are designed for the isolation and study of poly-ADP-ribosylated (PARylated) proteins. Through the use of this highly specific PAR affinity resin, PARylated proteins are isolated from cell or tissue lysates without the use of anti-PAR antibodies. The resin-bound proteins can be eluted from the affinity resin, and analyzed by immunoblotting or other methods. 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. The WWE Affinity Resin is a GST fusion protein of WWE PARbinding domain of human RNF146, amino acid residues 100-175, bound to a glutathione resin. The negative control resin is identical to the WWE Affinity Resin except for a single amino acid substitution that effectively abolishes PAR binding.

Description: WWE Affinity Resin (MBS376022) is highly purified GST-WWE fusion protein expressed in E. coli, and bound to glutathione beads. The WWE domain sequence is human RNF146 (Iduna) amino acid residues 100-175. It is useful for affinity purification (pulldown) of PARsylated proteins.

WWE Negative Control Resin (MBS376023 ) is identical to the WWE Affinity Resin resin except for a single amino acid substitution R163A, which abolishes PAR binding. The negative control resin is useful to control for non-specific binding, and its use is optional.

References

H.C. Kang et al. (2011) PNAS 108 14103 Y. Zhang et al. (2011) Nature Cell Biol. 13 623 M.G. Callow et al. (2011) PLoS ONE 6 e22595 S.A. Andrabi et al. (2011) Nature Medicine 17 692 Z.D. Zhou et al. (2011) Cell Adh. Migr. 5 463 Z. Wang et al. (2012) Genes & Dev. 26 235