WWE protein product blog
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The WWE n/a (Catalog #MBS376021) is a Protein and is intended for research purposes only. The product is available for immediate purchase. MyBioSource\'s WWE can be used in a range of immunoassay formats including, but not limited to, Western Blot (WB), Immunoblot (IB).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. Researchers should empirically determine the suitability of the WWE n/a for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process.
To buy or view more detailed product information and pricing, please click on the technical datasheet page below:
Please refer to the product datasheet for known applications of a given protein. We\'ve tested the WWE Affinity Resin Set with the following immunoassay(s):
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)
Our knowledge of the role of proteins in cellular processes is continually evolving. Most proteins, including WWE are typically involved in one or more signaling pathways or biological processes. Professionally manufactured recombinant proteins are increasingly becoming essential and commonplace tools for elucidating new knowledge about the role of proteins in both health and disease.
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 20�L 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 75�L 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) (20�L 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 500�L Lysis buffer.
6. 100�L 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.